Genetically modified human natural killer cell lines

ABSTRACT

The invention provides a natural killer cell, NK-92, modified to express an Fc receptor on the surface of the cell, such as CD16 (FcγRIII-A), or other Fcγ or Fc receptors. The modified NK-92 cell can be further modified to concurrently express an associated accessory signaling protein, such as FcεRI-γ, TCR-ζ, or to concurrently express interleukin-2 (IL-2) or other cytokines. Additional methods are disclosed for various assays, assessments, and therapeutic treatments with the modified NK-92 cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application60/586,581, filed Jul. 10, 2004, entitled A GENETICALLY MODIFIED HUMANNATURAL KILLER (NK) CELL LINE, the entirety of which is hereinincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with grants from the National Institutesof Health: NIH R01 CA083859 (NCI; 2000-2009), entitled “Negativesignaling by killer cell Ig-like receptors” and NIH R01 CA100226 (NCI;2004-2009), entitled “Mechanisms of NK cell activation by the KIR2DL4receptor.” The government may have certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

N/A

FIELD OF THE INVENTION

This invention relates generally to certain natural killer (NK) celllines that have been genetically engineered to express a cell surfacereceptor protein that participates in antibody-dependent cellularcytotoxicity responses. More specifically, the present invention relatesto a natural killer cell line NK-92 which, in a first embodiment, hasbeen modified to express an Fc cell surface receptor protein such asCD16, and in a second embodiment, has been modified to express both anFc cell surface receptor protein such as CD16 and one or more of theassociated accessory signaling proteins such as FcR-γ or TCR-ζ and/or acytokine such as IL-2.

BACKGROUND

A number of antibodies, most notably Rituximab (MabThera®;Hoffmann-LaRoche, Ltd; Basel, Switzerland) and Herceptin® (Genentech,Inc.; South San Francisco, Calif.), have shown significant therapeuticvalue as highly selective and effective anti-tumor agents. Althoughthese antibodies can bind to specific antigens on the tumor cells, theiranti-tumor activity depends at least in part on the subsequent bindingof natural killer (NK; a table of abbreviations is provided in Table Z,located after the Examples) cells to the Fc (constant) portion of theantibody with consequent destruction of the tumor cell via anantibody-dependent cellular cytotoxicity (ADCC) mechanism.

NK cells are a class of lymphocytes that typically compriseapproximately 10% of the lymphocytes in a human. The primary function ofNK cells is to provide an innate cellular immune response against tumorand infected (target) cells. Roles in the priming and regulation ofhumoral immune response, fetal development and the elimination ofstressed or damaged normal cells have also been demonstrated and/or areconsidered to be likely. NK cells, which are characterized as having aCD3⁻/CD56⁺ phenotype, display a variety of activating and inhibitorycell surface receptors. The binding or ligation of an activating NK cellreceptor to the corresponding ligand on a target cell triggers the NKcell to exert a cytotoxic effect directly against the target cell and tosecrete a variety of cytokines that perform functions such as thestimulation and recruitment of other elements of the immune system toact against the target. Activated NK cells lyse target cells via thesecretion of the enzymes perforin and granzyme, stimulation ofapoptosis-initiating receptors and other less well characterizedmechanisms.

NK cell inhibitory receptors predominantly engage with majorhistocompatibility complex class I (“MHC-I”) proteins on the surface ofa normal cell. When so engaged, these inhibitory receptors prevent NKcells from becoming activated. The MHC-I molecules define cells as“belonging” to a particular individual. As expression of these MHC-Imolecules can prevent NK cell activation toward a target cell, it isthought that NK cells can be activated only by cells on which these“self” MHC-I molecules are missing or defective, such as is often thecase for tumor or virus-infected cells. The NK cell phenotype andactivation pattern are distinct from that exhibited by cytotoxicT-lymphocytes (“CTLs,” CD3⁺/CD56⁻/CD8⁺ phenotype) that are activated bytarget cells that display small foreign peptide fragments derived fromviruses or tumor cells attached to the surface MHC-I molecules.Scientists have speculated that NK cells evolved as a response to tumorand infected cells that evade destruction by CTLs through suppression ordisruption of the display of peptide-presenting MHC-I molecules.

NK cells have been evaluated as a therapeutic agent in the treatment ofcertain cancers. The NK cells used for this purpose are isolated fromthe peripheral blood lymphocyte (“PBL”) fraction of blood from thesubject, expanded in cell culture in order to obtain sufficient numbersof cells, and then re-infused into the subject. Although the results ofthis therapy have been promising, preparation of the autologous NK cellsis expensive, labor intensive and time consuming. Furthermore, qualitycontrol of these cells is complicated by each preparation being subjectspecific. In particular, the quantity of NK cells that can be isolatedfrom a subject can vary substantially, and these cells are oftendeficient in proliferative ability and/or cytotoxic activity. Anotherlimitation on the use of NK cells as a therapeutic agent results fromthe presence of surface antigens on the cells that can evoke an immunerejection response when the cells are infused into a subject other thanthe one from which they were isolated. This necessitates careful MHC-Icross-matching between the donor and the recipient as well as the needto immuno-suppress the recipient.

The NK-like cell line NK-92 was discovered in the blood of a subjectsuffering from a non-Hodgkins lymphoma. NK-92 cells lack the majorinhibitory receptors that are displayed by normal NK cells, but retainthe majority of the activating receptors. Characterization of the NK-92cell line (Gong et al., 1994; Yan et al., 1998) revealed that NK-92cells are cytotoxic to a significantly broader spectrum of tumor andinfected cell types than are NK cells, and further that they oftenexhibit higher levels of cytotoxicity toward these targets. NK-92 cellsdo not, however, attack normal cells nor do they elicit an immunerejection response. In addition, NK-92 cells can be readily and stablygrown and maintained in continuous cell culture and, thus, can beprepared in large quantities under c-GMP compliant quality control. Thiscombination of characteristics has resulted in NK-92 being entered intopresently on-going clinical trials for the treatment of multiple typesof cancers.

Although NK-92 cells retain almost all of the activating receptors andcytolytic pathways associated with NK cells, they do not express theCD16 receptor and, therefore, cannot lyse target cells via the ADCCmechanism. This means that despite their other benefits, NK-92 cellscannot potentiate the anti-tumor and anti-infection effects ofendogenous or exogenous antibodies in the manner of NK cells. OtherNK-like cell lines in addition to NK-92 are also known. Some of theseother NI-like cell lines express CD16, but this expression is unstable;the cells are typically difficult to grow in cell culture; and fewexhibit robust cytotoxic activity. For these reasons, only NK-92 of thecurrently known NK-like cell lines is a viable candidate as atherapeutic agent even though it lacks CD16 and, consequently, theability to kill target cells via the ADCC mechanism.

Thus, it would be an advantage to restore CD16 expression and theability to act via the ADCC mechanism to NK-92 cells, thus permittingthose cells to be used in concert with antibodies for therapeutic andrelated purposes. However, NK cell lines have been found to berecalcitrant to gene transfer, a feature that has hampered thedevelopment of such cell lines for research or therapeutic purposes. ForNK-92 cells, transformation efficiencies of only 5-15% and 10-20% havebeen achieved using particle-mediated gene transfer or retroviraltransduction (Nagashima et al., 1998; Tam et al., 1999). NK-92 celllines that stably expresses the CD16 cell surface receptor are currentlyunavailable.

SUMMARY

The various embodiments of the invention provide or utilize an NK-92cell modified to express a Fc receptor such as CD16 (FcγRIII-A), or moregenerally, any other Fc receptor, on the surface of the cell.

In a first aspect, the invention is directed to NK-92 cells modified toexpress a Fc receptor on a surface of the cell; the Fc receptor can bean activating Fcγ receptor, CD16 (FcγRIII-A), or any member of an Fcreceptor class, such as FCγRI (CD64), FCγRII (CD32), FCγRIII, FcRn, Fcαand Fcε. The Fc receptors can be of any binding affinity for theirligands, or fragments of their ligands, including low- and high-bindingaffinity forms. The NK-92 cells can be modified by introducing apolynucleotide that encodes a polypeptide having at least 70%, 80%, 90%,95%, 99% or 100% identity to the amino acid sequences of SEQ ID NO:1 orSEQ ID NO:2; one such polynucleotide includes SEQ ID NO:3. The NK-92cells can be further modified to express one or more associatedaccessory signaling polypeptides, cytokines, or fragments thereof; suchexpression can correlate with increased surface expression of the Fcreceptor. Associated accessory signaling polypeptides include FcεRI-γ(SEQ ID NO:5) or TCR-ζ (SEQ ID NO:7). Expression of a cytokine (such asinterleukin-2) can also correlate with viability or cytotoxicity of themodified NK-92 cells.

In a second aspect, the invention is directed to methods for in-vitroassessment of the efficacy of an antibody to induce cell death. Suchmethods can include the steps of exposing a target cell to an antibody(monoclonal (purified or in hybridoma supernates), polyclonal, chimeric(such as one having at least two dissimilar antigen binding domains(wherein one binding domain can be adapted to bind the Fc receptor), orany other form of antibody), exposing the target cell to a modifiedNK-92 cell expressing an Fc receptor; and then monitoring the targetcell for cytotoxicity, cytolysis, or apoptosis, or a combinationthereof. Pluralities of cells and antibodies can be used. The targetcells used in the methods can have a lysis or aptotic rate of about5%-30% in the presence of the modified NK-92 cells in the absence ofantibody. Effector:target ratios include 0.5:1 to 100:1, including 1:1and 20:1. Target cells in the methods include SKOV-3, P815, THP-1,U373MG, T98G, A ML193, SR91, ALL1, and REH; these and any other targetcells can be modified to increase expression of the antigen to which theantibody binds. Appropriate negative controls include using unmodifiedNK-92 cells.

The NK-92 cells in this aspect can include those modified to express aFc receptor on a surface of the cell; the Fc receptor can be anactivating Fcγ receptor, CD16 (FcγRIII-A), or any member of an Fcreceptor class, such as FCγRI (CD64), FCγRII (CD32), FCγRIII, FcRn, Fcαand Fcε. The Fc receptors can be of any binding affinity for theirligands, or fragments of their ligands, including low- and high-bindingaffinity forms. The NK-92 cells can be modified by introducing apolynucleotide that encodes a polypeptide having at least 70%, 80%, 90%,95%, 99% or 100% identity to the amino acid sequences of SEQ ID NO:1 orSEQ ID NO:2; one such polynucleotide includes SEQ ID NO:3. The NK-92cells can be further modified to express one or more associatedaccessory signaling polypeptides, cytokines, or fragments thereof; suchexpression can correlate with increased surface expression of the Fcreceptor. Associated accessory signaling polypeptides include FcεRI-γ(SEQ ID NO:5) or TCR-ζ (SEQ ID NO:7). Expression of a cytokine (such asinterleukin-2) can also correlate with viability or cytotoxicity of themodified NK-92 cells. Cytokines can also be added to the assay fromexogenous sources.

In a third aspect, the invention is directed to methods for detectingcytolytic and apoptosis-inducing activity, the method including thesteps of exposing a target cell in the absence of antibodies to a NK-92cell expressing an Fc receptor, and then monitoring the target cell forcytotoxicity, cytolysis or apoptosis. Monitoring can include determiningIFN-γ or cytokine expression levels. The method can further includeapplying blocking agents, such as activating receptor-masking antibodiesor polypeptides (or fragments of these) to suppress one or moreactivating receptors on the NK-92 cell.

In a fourth aspect, the invention is directed to methods of assaying theefficacy of an antibody to treat a tumor, infection or other lesion, themethod including the steps of administering an antibody (or plurality ofantibodies) to a subject; administering modified NK-92 cells expressingan Fc receptor to the subject; and then monitoring the tumor, infectionor lesion. The efficacy of the antibody in the treatment correlates withsuppression of the tumor, infection or lesion in the subject. Monitoringcan include determining IFN-γ or cytokine expression levels. The methodcan further include applying blocking agents, such as activatingreceptor-masking antibodies or polypeptides (or fragments of these) tosuppress one or more activating receptors on the NK-92 cell.

The antibody can be monoclonal (purified or in hybridoma supernates),polyclonal, chimeric (such as one having at least two dissimilar antigenbinding domains (wherein one binding domain can be adapted to bind theFc receptor), or any other form of antibody. Cytokines (such asinterleukin-2), or fragments thereof, can be expressed from modifiedNK-92 cells or supplied exogenously. Subjects include bovines (e.g.,cows), swine (e.g., pigs, hogs), rabbits, alpacas, horses, canines(e.g., dogs), felines (e.g., cats), ferrets, rats, mice, fowl (chickens,turkeys) and buffalo. Subjects can also be human.

The NK-92 cells in this aspect can include those modified to express aFc receptor on a surface of the cell; the Fc receptor can be anactivating Fcγ receptor, CD16 (FcγRIII-A), or any member of an Fcreceptor class, such as FCγRI (CD64), FCγRII (CD32), FCγRIII, FcRn, Fcαand Fcε. The Fc receptors can be of any binding affinity for theirligands, or fragments of their ligands, including low- and high-bindingaffinity forms. The NK-92 cells can be modified by introducing apolynucleotide that encodes a polypeptide having at least 70%, 80%, 90%,95%, 99% or 100% identity to the amino acid sequences of SEQ ID NO:1 orSEQ ID NO:2; one such polynucleotide includes SEQ ID NO:3. The NK-92cells can be further modified to express one or more associatedaccessory signaling polypeptides, cytokines, or fragments thereof; suchexpression can correlate with increased surface expression of the Fcreceptor. Associated accessory signaling polypeptides include FcεRI-γ(SEQ ID NO:5) or TCR-ζ (SEQ ID NO:7).

In yet another, fifth aspect, the invention is directed to methods oftreating a subject, the subject having a tumor, infection or otherlesion, the method including administering to a subject antibodies thatspecifically bind to the tumor, infection or other lesion; and thenadministering to the subject modified NK-92 cells expressing an Fcreceptor. A reduction in the tumor, infection or lesion indicates atherapeutic response. Monitoring can include determining IFN-γ orcytokine expression levels. The method can further include applyingblocking agents, such as activating receptor-masking antibodies orpolypeptides (or fragments of these) to suppress one or more activatingreceptors on the NK-92 cell.

The antibody can be monoclonal (purified or in hybridoma supernates),polyclonal, chimeric (such as one having at least two dissimilar antigenbinding domains (wherein one binding domain can be adapted to bind theFc receptor), or any other form of antibody. Cytokines (such asinterleukin-2), or fragments thereof, can be expressed from modifiedNK-92 cells or supplied exogenously. Subjects include bovines (e.g.,cows), swine (e.g., pigs, hogs), rabbits, alpacas, horses, canines(e.g., dogs), felines (e.g., cats), ferrets, rats, mice, fowl (chickens,turkeys) and buffalo. Subjects can also be human.

The NK-92 cells in this aspect can include those modified to express aFc receptor on a surface of the cell; the Fc receptor can be anactivating Fcγ receptor, CD16 (FcγRIII-A), or any member of an Fcreceptor class, such as FCγRI (CD64), FCγRII (CD32), FCγRIII, FcRn, Fcαand Fcε. The Fc receptors can be of any binding affinity for theirligands, or fragments of their ligands, including low- and high-bindingaffinity forms. The NK-92 cells can be modified by introducing apolynucleotide that encodes a polypeptide having at least 70%, 80%, 90%,95%, 99% or 100% identity to the amino acid sequences of SEQ ID NO:1 orSEQ ID NO:2; one such polynucleotide includes SEQ ID NO:3. The NK-92cells can be further modified to express one or more associatedaccessory signaling polypeptides, cytokines, or fragments thereof; suchexpression can correlate with increased surface expression of the Fcreceptor. Associated accessory signaling polypeptides include FcεRI-γ(SEQ ID NO:5) or TCR-ζ (SEQ ID NO:7).

Numerous other advantages and features of the present invention willbecome readily apparent from the following detailed description of theinvention and the embodiments thereof, from the claims and from theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, features and advantages of the present invention will bemore readily appreciated upon reference to the following disclosure whenconsidered in conjunction with the accompanying drawings.

FIG. 1 shows flow cytometer scatter diagrams of NK-92 cells transducedwith CD16 cDNA using the pBMN-IRES-EGFP vector after staining with noprimary antibody (FIG. 1A) and with both primary (anti-CD16) andsecondary antibody (FIG. 1B). EGFP expression is assessed on the x-axis,and surface CD16 expression is on the y-axis.

FIG. 2 shows flow cytometer scatter diagrams showing the expression ofCD16 by NK-92 cells transduced with CD16 alone (FIG. 2A), and theincrease in CD16 expression when NK-92 cells are transduced with CD16cDNA in combination with FcεRI-γ cDNA (γ; in pBMN-IRES-EGFP vector)(FIG. 2B), or CD3 ζ cDNA (FIG. 2C).

FIG. 3 is a graphical diagram showing redirected cytotoxicity ofFcγRII/III⁺ P815 target cells by NK-92-CD16 cells induced by anti-CD16antibody (3G8), but not antibodies toward CD56 (B159) or KIR (DX9).Cells were assayed using ⁵¹Cr release from P815 target cells at theindicated effector to target ratios.

FIG. 4 is a graphical diagram illustrating redirected cytotoxicity ofFcγRII/III⁺ THP-1 target cells by NK-92-CD16 cells (filled symbols)induced by anti-CD16 antibody (3G8; squares), but not anti-NKR-P1antibody (B199; triangles). Redirected cytotoxicity was not induced byanti-CD16 in NK-92 cells transduced with mouse IgM cDNA (open symbols).

FIG. 5 is a graphical diagram illustrating redirected cytotoxicity ofSKOV-3 target cells by NK-92-CD16 cells (triangles), but not mouseIgM-transduced NK-92 (squares), induced by bi-specific 2B1 antibody(filled symbols). 2B1 contains F(ab) domains recognizing both Her2/neuantigen on SKOV-3 cells and CD16 on NK-92-CD16.

FIG. 6 is a graphical diagram illustrating redirected cytotoxicity ofNoGFP NK-92-CD16, NK-92-CD16-γ, and NK-92-CD16-ζ cells against P815target cells in combination with the indicated concentration of 2B1chimeric bi-specific monoclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention is susceptible of embodiment in manydifferent forms, there are shown in the drawings, and will be describedherein in detail specific embodiments and examples thereof, with theunderstanding that the present disclosure is to be considered as anexemplification of the principles of the invention and is not intendedto limit the invention to the specific embodiments illustrated.

The present invention is directed towards cell lines and methods thatpotentiate and broaden the effective scope of the ADCC response. Thepresent invention provides an NK-92 cell line that stably expresses anFc cell surface receptor protein, such as CD16. (Several differentnomenclatures have evolved to refer to certain Fc receptors; they areused interchangeably herein, including CD16, FCγRIII-A, and theirpolymorphisms or other forms having varied affinity levels).

Many biotechnology companies are presently developing novel monoclonalantibodies for use in cancer immuno-therapies. The intent of thisdevelopment effort is to prepare antibodies that bind to particularprotein antigens that are uniquely expressed on the surfaces of specifictypes of tumor cells. Cytotoxic NK cells can, in turn, bind to the Fc(constant) region of these tumor-bound antibodies via the CD16 receptorsdisplayed on the NK cells and initiate lysis of the tumor cell throughthe ADCC mechanism. The high efficacy and specificity of therapeuticagents based upon this approach has been clinically demonstrated throughthe use of the monoclonal antibody Herceptin (anti-ErbB2) for thetreatment of ErbB2-expressing breast carcinomas and the use of themonoclonal antibody Rituximab (anti-CD20) for the treatment of B-celllymphomas. Numerous other therapeutic antibodies that target a widevariety of additional tumor-specific antigens are under development.

An essential part of the development of antibody-based cancer therapiesis the in-vitro determination of the efficacy and specificity of NKcell-mediated ADCC that is imparted by the binding of the antibody tothe target tumor cell. These tests are typically conducted usingperipheral blood lymphocytes (PBL) obtained from normal human donors orNK cells isolated from the PBL blood fraction as effector cells. Inaddition to the cumbersome burden inherent in routinely obtaining andprocessing human blood samples, there is considerable variability in theactivity and quantity of effector cells obtained from different donorsand similar variability between samples obtained from the same donor.Part of this variability is intrinsic and arises from the polyclonalnature of NK cells. In particular, allelic variations in theextracellular portion of the CD16 gene can result in significantdifferences in CD16 affinity for the Fc portions of antibodies anddifferences in the activation status of donor NK cells can alter thelevel of CD16 expression on the cell surface. The concentration of NKcells in the PBL fraction also varies, partly for genetic reasons andpartly as a reflection of the physiological status of the donor. Thiseffector cell variability greatly complicates the assessment ofmonoclonal antibodies as potential therapeutic agents. Furthermore,although NK cells expressing the low binding affinity form of CD16 aremost common, the high affinity isotype is sufficiently common thatantibody testing needs to be carried using NK cells of both forms (Koeneet al., 1997). Identifying donors homozygous for low and high affinityallelic forms of CD16 is a difficult task. The availability of a clonalhuman NK cell population that expresses a consistent level of CD16activity would provide substantial benefit as a standard effector in theevaluation of antibodies for ADCC activity and specificity.

In like manner, antibody-based therapies can benefit from the presenceof NK cells having known high levels of Fc-binding capacity andcytotoxic activity within the subject. In addition to the variabilitiespreviously described, NK cells isolated from cancer subjects are oftenfound to have been rendered defective, deficient or ineffective byactions of the tumor cells. Some types of tumor cells, for example, areable to kill or deactivate NK cells in a subject-specific manner. Othertypes of tumor cells similarly are able to interfere with NK cellproduction, activity and/or specificity. Such variability makes relianceon subject NK cells problematic in a therapeutic setting and suggeststhat the co-administration of known quantities of exogenous NK cellshaving a known level of activity along with an appropriate antibody canresult in more consistent therapeutic effects. Again, the availabilityof a clonal human NK cell population that expresses a consistent levelof CD16 activity is expected to provide substantial benefit.

One major limitation on the usefulness of NK cells is that they expressa repertoire of cell surface receptors that, when bound to thecorresponding MHC-I ligand on a normal target cell, strongly andspecifically prevent destruction of the target cell by inhibiting NKcell activation, cytotoxicity and cytokine response. This inhibition canabrogate the activation of NK cells caused by the binding of NK cellactivating receptors to their conjugate ligands on the target cell. Thusa NK cell will destroy a target cell that displays only activatingligands, but will spare a target cell that displays MHC-I inhibitoryligands even if activating ligands are also present.

Almost all cells in mammals display certain polymorphic cell surfaceproteins of the MHC-I on their surfaces. These MHC-I proteins have theprimary function of displaying peptide antigens that are fragmentsderived from proteins expressed within a cell and are classified asbeing MHC-I complexes. The MHC complexes displayed on normal cells areunique to each individual and are often referred to as being markers of“self” for that individual. Exogenous cells introduced bytransplantation display MHC molecules and associated peptides thatdiffer from those of the host individual and are therefore referred toas being “non-self.” Non-self MHC-peptide complexes can also appear onaberrant cells as a result of processes that alter the peptidespresented on the MHC molecules. In particular, non-self peptides aredisplayed by many, but not all, tumor cells and infected cells.

NK cell inhibitory receptors consist of several families of proteinsthat recognize, bind to and are triggered to send “negativeintracellular signals” by encountering intact self MHC-I proteins on thesurface of a normal self, target cell. NK cells are therefore preventedfrom attacking and destroying normal cells that display the appropriateself MHC-I constellation. Tumor and infected cells that display thisself MHC-I constellation are also immune from attack. Only cells thatdisplay non-self or no MHC-I are subject to destruction by NK cells.Some types of tumor and infected cells that do not display self MHC-Icomplexes have, however, evolved mechanisms that allow them to escapethis fate. Examples of such escape mechanisms include expression ofsurrogate MHC-1-like, NK cell inhibitory receptor ligands and thesecretion of soluble ligands that suppress NK cell functions. Tumor andinfected cells that implement such escape mechanisms are refractory toNK cell-mediated lysis. The efficacy of NK cells as an agent for thedestruction of cancer cells is therefore limited by the presence ofappropriate MHC-I ligands for NK cell inhibitory receptors. Theavailability of a human NK cell population that does not express NK cellinhibitory receptors would beneficially expand the range of cancers andinfections that can be treated using antibody-based therapeutic agents.

NK-92 is a NK-like cell line that was initially isolated from the bloodof a subject suffering from a large granular lymphoma and subsequentlypropagated in cell culture. The NK-92 cell line has been described (Gonget al., 1994; Klingemann, 2002). NK-92 cells have a CD3−/CD56+ phenotypethat is characteristic of NK cells. They express all of the known NKcell-activating receptors except CD16, but lack all of the known NK cellinhibitory receptors except NKG2A/CD94 and ILT2/LIR1, which areexpressed at low levels. Furthermore, NK-92 is a clonal cell line that,unlike the polyclonal NK cells isolated from blood, expresses thesereceptors in a consistent manner with respect to both type and cellsurface concentration. Similarly, NK-92 cells are not immunogenic and donot elicit an immune rejection response when administeredtherapeutically to a human subject. Indeed NK-92 cells are welltolerated in humans with no known detrimental effects on normal tissues.While NK-92 cells, unlike NK cells and cells of most of the other knownNK-like cell lines, have been engineered to express novel proteins bymeans of transduction using retroviral vectors (Campbell et al., 2004;Kikuchi-Maki et al., 2003; Klingemann, 2002; Yusa and Campbell, 2003;Yusa et al., 2002; Yusa et al., 2004), such engineering has proveddifficult as evidenced by numerous failures to engineer NK-92 cells toexpress an Fc receptor. More particularly, despite the clear potentialbenefits which could be anticipated from an NK-92 cell line modified toexpress CD16, such genetic modification had not been achieved in factuntil the present invention.

This unique combination of characteristics renders NK-92 as a suitableplatform upon which the present invention can be constructed. Inparticular, the lack of inhibitory receptors means that NK-92 cells arenot MHC-restricted and can act effectively against any cell thatdisplays an appropriate activating ligand independent of any MHC-Iinhibitory ligands that can also be expressed. The lack ofimmunogenicity coupled with the relative ease with which NK-92 cells canbe grown in culture means that they can be prepared in bulk andadministered to any subject as the need arises. The stability andconsistency of NK-92 cells males them suitable for use as a referencematerial and therapeutic agent. In addition, the present inventionprovides the ability to transduce NK-92 cells with genes for novelproteins, such as Fc receptors, in conjunction with the ability of NK-92cells to stably express these proteins, as explained in greater detailbelow.

The use of NK-92 cells as a therapy for cancers is currently beingevaluated with promising results in human clinical trials. The benefitsof NK-92 cells are being further exploited through the development ofgenetically engineered NK-92 variants that express a protein constructthat covalently links an antibody-like binding domain to a signalingdomain such as TCR-ζ (Genbank Accession No. J04132; SEQ ID NO:6) (TCR-ζ(Genbank Accession No. J04132; SEQ ID NO:6)) (Klingemann, 2002; Maki etal., 2001; Uherek et al., 2001; Uherek et al., 2002). In theseconstructs, the antibody-like domain is structured to specifically bindto an antigen that is expressed by a target cell while the signalingdomain is one that is known to trigger NK and NK-92 cell activity whenstimulated. It has, to date, been demonstrated in-vitro and in animalmodels that the binding of the antibody-like domain of such a constructto its antigen on a target cell triggers the NK-92 cell in a manner suchthat it rapidly and efficiently destroys the target cell via directconjugation. The utility of these constructs as therapeutic agents is,however, limited by the need to design, prepare and validate a uniqueconstruct for each specific type of cancer or infection to be treated.The availability of a single NK-92 cell variant that can be used totreat a broad range of cancers and infections is of beneficial utility.

One area in which NK-92 cells can be improved pertains to the use ofNK-92 cells in subjects, in that the cytotoxicity, cell surface receptorconcentration and survival of NK-92 cells as well as the range of tumorand infected cell types that are attacked have been shown to beincreased by the presence of low concentrations of the cytokineinterleukin-2 (IL-2). The cost of the exogenously added IL-2 needed tomaintain and expand NK-92 cells in commercial scale culture issignificant, while the administration of IL-2 to human subjects insufficient quantity to achieve the desired effects is also known tocause adverse side effects. This limitation has been addressed by thedevelopment of the IL-2 secreting NK-92 ml and NK-92ci cell lines byretroviral transduction of NK-92 cells with the gene for IL-2(Klingemann, 2002; Nagashima et al., 1998). The levels of IL-2 secretedby these cell lines are sufficient to optimize NK-92 cell survival andactivity, but are below the level generally associated with the onset ofadverse side effects.

Another area in which NK-92 cells can be improved, and the focus of thepresent invention, concerns the fact that an unmodified NK-92 cell doesnot express CD16 and therefore is ineffective in killing target cellsvia the ADCC mechanism. Although NK-92 cells are widely used as a modelsystem for the study of NK cell activation, action and inhibition, thelack of CD16 expression precludes the use of NK-92 cells for theevaluation of efficacy of antibodies as therapeutic agents and the useof NK-92 cells as a therapeutic agent that is co-administered with anantibody. The present invention addresses this limitation by causingNK-92 cells to express CD16. Additional utility and benefit of thepresent invention will become apparent in the following descriptions.

Modifying NK-92 Cells

CD16 is most commonly found in a form that has a relatively low bindingaffinity for the Fc portion of IgG molecules. An alternative form thatexhibits a higher binding affinity is found in some individuals. The lowand high affinity forms of CD16 differ only by the substitution ofvaline (high affinity) for phenylalanine (low affinity) at position 157in the polypeptide chain. The complete sequences of the low and highaffinity forms can be found in the SwissProt database as entries P08637(SEQ ID NO:1) and VAR_(—)008801 (SEQ ID NO:2), respectively and arepresented in Tables 1 and 2; the polynucleotide encoding SEQ ID NO:1(SEQ ID NO:3) is presented in Table 3.

CD16 was introduced into NK-92 cells by means of retroviral transductionin the following manner. Complementary DNA encoding the gene for eitherthe low or high affinity form of CD16 was sub-cloned into a bi-cistronicretroviral expression vector, pBMN-IRES-EGFP (obtained from G. Nolan,Stanford University, Stanford, Calif.) using the BamHI and NotIrestriction sites in accordance with standard methods (e.g., (Ausubel,2002; Sambrook and Russell, 2001)). This expression vector was thentransfected into the Phoenix-Amphotropic retroviral packaging cell lineand the resulting virus-containing supernate was used to transduce NK-92cells, although alternative methods including those in which vectorsincorporate EGFP or other fluorescent proteins (yellow, red, cyan, etc.)can be used without departing from the spirit and scope of thisinvention, and the Phoenix-Amphotropic retroviral packaging cell linewere both obtained from and are available to the public from the LelandStanford University, Stanford, Calif., USA; (Kinsella and Nolan, 1996;Nolan and Kinsella, 1998)). As indicated below, other vectors andpackaging cell lines, both those currently known and those which can bedeveloped in the future, can be used. Transduced NK-92 cells expressingCD16 on their surface (NK-92-CD16, also known as CD16/FcεRIγ-NK-92) wereseparated from the residual non-transduced NK-92 cells using afluorescence activated cell sorter (FACS). When appropriate to theintended use, the NK-92-CD16 cells were further sub-sorted on the basisof CD16 expression level using a FACS, based upon coordinate expressionof Enhanced Green Fluorescent Protein (EGFP). The resulting NK-92-CD16cells stably express CD16 in cell culture without the need for periodicantibiotic selection.

TABLE 1 Polypeptide seciuence for SEQ ID NO: 1 (Low affinityimmunoglobulin gamma Fc region receptor III-A [Precursor]) Met Trp GlnLeu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1               5                   10                  15 Gly Met ArgThr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro            20                  25                  30 Gln Trp Tyr ArgVal Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln        35                  40                  45 Gly Ala Tyr Ser ProGlu Asp Asn Ser Thr Gln Trp Phe His Asn Glu    50                  55                  60 Ser Leu Ile Ser Ser GlnAla Ser Ser Tyr Phe Ile Asp Ala Ala Thr65                  70                  75                  80 Val AspAsp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                85                  90                  95 Ser Asp ProVal Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln            100                 105                 110 Ala Pro Arg TrpVal Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys        115                 120                 125 His Ser Trp Lys AsnThr Ala Leu His Lys Val Thr Tyr Leu Gln Asn    130                 135                 140 Gly Lys Gly Arg Lys TyrPhe His His Asn Ser Asp Phe Tyr Ile Pro145                 150                 155                 160 Lys AlaThr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe                165                 170                 175 Gly Ser LysAsn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln            180                 185                 190 Gly Leu Ala ValSer Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln        195                 200                 205 Val Ser Phe Cys LeuVal Met Val Leu Leu Phe Ala Val Asp Thr Gly    210                 215                 220 Leu Tyr Phe Ser Val LysThr Asn Ile Arg Ser Ser Thr Arg Asp Trp225                 230                 235                 240 Lys AspHis Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys                245                 250

TABLE 2 Polypeptide sequence for SEQ ID NO: 2 (Low affinity variant 157F-> V immunoglobulin gamma Fc region receptor III-A [Precursor]) Met TrpGln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala1               5                   10                  15 Gly Met ArgThr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro            20                  25                  30 Gln Trp Tyr ArgVal Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln        35                  40                  45 Gly Ala Tyr Ser ProGlu Asp Asn Ser Thr Gln Trp Phe His Asn Glu    50                  55                  60 Ser Leu Ile Ser Ser GlnAla Ser Ser Tyr Phe Ile Asp Ala Ala Thr65                  70                  75                  80 Val AspAsp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu                85                  90                  95 Ser Asp ProVal Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln            100                 105                 110 Ala Pro Arg TrpVal Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys        115                 120                 125 His Ser Trp Lys AsnThr Ala Leu His Lys Val Thr Tyr Leu Gln Asn    130                 135                 140 Gly Lys Gly Arg Lys TyrPhe His His Asn Ser Asp Val Tyr Ile Pro145                 150                 155                 160 Lys AlaThr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe                165                 170                 175 Gly Ser LysAsn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln            180                 185                 190 Gly Leu Ala ValSer Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln        195                 200                 205 Val Ser Phe Cys LeuVal Met Val Leu Leu Phe Ala Val Asp Thr Gly    210                 215                 220 Leu Tyr Phe Ser Val LysThr Asn Ile Arg Ser Ser Thr Arg Asp Trp225                 230                 235                 240 Lys AspHis Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys                245                 250

TABLE 3 Polynucleotide sequence (mRNA) for SEQ ID NO: 3 (Low affinityimmunoglobulin gamma Fc region receptor III-A [Precursor]) atgtggcagctgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcccaaaggctgt ggtgttcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtgactctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatgagagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgacagtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaagtccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagaccctattcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacagaatggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacactcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540 gtgtcttcagagactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600 tcattctttccacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660 gtggacacaggactatattt ctctgtgaag acaaacattc gaagctcaac aagagactgg 720 aaggaccataaatttaaatg gagaaaggac cctcaagaca aatga 765

Recombinant Expression Vectors and Host Cells

Vectors are tools used to shuttle DNA between host cells or as a meansto express a polynucleotide sequence. Inserting the DNA of interest,such as a CD16 sequence or a fragment, is accomplished by ligationtechniques and/or mating protocols well known to the skilled artisan.Such DNA is inserted such that its integration does not disrupt anynecessary components of the vector. In the case of vectors that are usedto express the inserted DNA as a polypeptide, the introduced DNA isoperably-linked to the vector elements that govern its transcription andtranslation.

Vectors can be divided into two general classes: Cloning vectors arereplicating plasmid or phage with regions that are non-essential forpropagation in an appropriate host cell, and into which foreign DNA canbe inserted; the foreign DNA is replicated and propagated as if it werea component of the vector. An expression vector (such as a plasmid,yeast, or animal virus genome) is used to introduce foreign geneticmaterial into a host cell or tissue in order to transcribe and translatethe foreign DNA. In expression vectors, the introduced DNA isoperably-linked to elements, such as promoters, that signal to the hostcell to transcribe the inserted DNA. Some promoters are exceptionallyuseful, such as inducible promoters that control gene transcription inresponse to specific factors. Operably-linking a CD16 polynucleotide toan inducible promoter can control the expression of a CD16 gene orfragments. Examples of inducible promoters include those that aretissue-specific, which relegate expression to certain cell types,steroid-responsive (e.g., glucocorticoids (Kaufman, 1990) andtetracycline), or heat-shock reactive. Some bacterial repressionsystems, such as the lac operon, have been exploited in mammalian cellsand transgenic animals (Fieck et al., 1992; Wyborski et al., 1996;Wyborski and Short, 1991). Other desirable inducible promoters includethose that are not endogenous to the cells in which the construct isbeing introduced, but, however, are responsive in those cells when theinduction agent is exogenously supplied.

Vectors have many manifestations. A “plasmid” is a circular doublestranded DNA molecule that can accept additional DNA fragments. Viralvectors can also accept additional DNA segments into the viral genome.Certain vectors are capable of autonomous replication in a host cell(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) integrate into the genome of a host cell and replicate as partof the host genome. In general, useful expression vectors are plasmidsand viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses); other expression vectors canalso be used.

Vector choice is dictated by the organisms or cells being used and thedesired fate of the vector. Vectors can replicate once in the targetcells, or can be “suicide” vectors. In general, vectors comprise signalsequences, origins of replication, marker genes, enhancer elements,promoters, and transcription termination sequences. Vectors often use aselectable marker to facilitate identifying those cells that haveincorporated the vector. Table F summarizes many of the availablemarkers.

“Host cell” and “recombinant host cell” are used interchangeably. Suchterms refer not only to a particular subject cell but also to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the term.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are well known in the art (see examples in Table B). Thechoice of host cell dictates the preferred technique for introducing thepolynucleotide of interest. Introduction of polynucleotides into anorganism may also be done with ex vivo techniques that use an in vitromethod of transfection, as well as established genetic techniques, ifany, for that particular organism. Such procedures can similarly beemployed for the transduction of genetically engineered NK-92 cells,including NK-92-CD16-γ, NK-92-CD16-ζ, NK-92 ml and NK-92ci. The NK-92,NK-92 ml and NK-92ci cell lines are deposited with the American TypeCulture Collection (ATCC, 10801 University Boulevard, Manassas, Va.20110-2209) under Deposit Numbers CRL-2407 (deposited to generaldepository on Sep. 3, 1998; transferred to patent depository on Apr. 11,2005 and assigned Deposit No. PTA-6670), CRL-2408 and CRL-2409,respectively. No-GFP-CD16.NK-92 cell line (high affinity; 176V; SEQ IDNO:2) was deposited with ATCC on Sep. 9, 2005 and was assigned AccessionNo. PTA-6967. GFP-CD16-F/F.NK-92 (low affinity; SEQ ID NO:1) andGFP-CD16-V/V.NK-92 cell line (high affinity; SEQ ID NO:2) were depositedwith ATCC on Dec. 13, 2007. Viability testing In conjunction withAmendment and Response to Non-Final Office Action was completed, and aCertificate of Deposit was issued by the ATCC on Feb. 14, 2008, for thelow affinity cell line which was assigned ATCC Patent DepositDesignation No. PTA-8837. Viability testing was completed, and aCertificate of Deposit was issued by the ATCC on Feb. 13, 2008, for thehigh affinity cell line which was assigned ATCC Patent DepositDesignation No. PTA-8836.

TABLE A Methods to introduce polynucleotide into cells Cells MethodsReferences Notes Mammalian Calcium phosphateN-(2-Hydroxyethyl)piperazine- Cells may be “shocked” with cellstransfection N′-(2-ethanesulfonic acid glycerol or dimethylsulfoxide(HEPES) buffered saline solution (DMSO) to increase transfection (Chenand Okayama, 1988; Graham efficiency (Ausubel, 2002). and van der Eb,1973; Wigler et al., 1978) BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffered solution (Ishiura et al., 1982)Diethylaminoethyl (Fujita et al., 1986; Lopata Most useful fortransient, but (DEAE)-Dextran et al.,1984; Selden et al., not stable,transfections. transfection 1986) Chloroquine can be used to increaseefficiency. Electroporation (Neumann et al., 1982; Especially useful forhard-to- Potter, 1988; Potter et al., transfect lymphocytes. 1984; Wongand Neumann, 1982) Cationic lipid (Elroy-Stein and Moss, 1990; reagenttrans- Felgner et al., 1987; Applicable to both fection. Rose et al.,1991; Whitt et al., in vivo and in vitro 1990) transfection RetroviralProduction exemplified by Lengthy process, many packaging (Cepko et al.,1984; lines available at ATCC. Applicable Miller and Buttimore, 1986; toboth in vivo and in vitro Pear et al., 1993) Infection transfection. invitro and in vivo: (Austin and Cepko, 1990; Bodine et al., 1991; Feketeand Cepko, 1993; Koehne et al., 2003; Lemischka et al., 1986; Turner etal., 1990) Polybrene (Chaney ei al., 1986; Kawai and Nishizawa, 1984)Microinjection (Capecchi, 1980) Can be used to establish cell linescarrying integrated copies of DFF DNA sequences. Protoplast(Rassoulzadegan et al., 1982; fusion Sandri-Goldin et al., 1981;Schaffner, 1980)

Other vectors and packaging cell lines have been used in the preparationof genetically modified variants of NK-92 cells (Klingemann, 2002;Nagashima et al., 1998; Tam et al., 1999; Uherek et al., 2002) and canbe used equivalently herein. Retroviral transduction systems other thanthose of the Examples discussed below have also been successfully usedto transduce a variety of genes into NK-92 cells. By way of example,these alternative methods include, but are not limited to the p-JETvector in conjunction with FLYA13 packaging cells (Gerstmayer et al.,1999), the plasmid-based kat retroviral transduction system, andDFG-hIL-2-neo/CRIP (Nagashima et al., 1998). Electroporation and “genegun” introduction of the vector into the packaging cells is alsopracticed. Use of the pBMN-IRES-EGFP vector in combination with thePhoenix-Amphotropic packaging cell line is convenient for the purpose ofthis and the following Examples in that it provides high efficiencies ofPhoenix-Amphotropic cell transfection; the use of Moloney LTR promotersresults in a high level of CD16 expression; the virus is produced athigh titers; the efficiency of NK-92 transduction is improved over othervectors that have been used to transduce NK-92; and the vector providesadequate space to accommodate the CD16 cDNA or alternative inserts. ThepBMN-IRES-EGFP vector further incorporates genes for enhanced greenfluorescent protein (EGFP), which can be used as an endogenous surrogatemarker for gene expression. The Phoenix cell line stably expresses thisvector in episomal form along with producing other viral components,thus allowing the cells to stably produce virus for an extended periodof time. Importantly, previously described publications (Klingemann,2002; Nagashima et al., 1998; Tam et al., 1999; Uherek et al., 2002)have established that alternative retroviral systems can be used tointroduce cDNAs into NK-92 without departing from the spirit and scopeof the invention.

A “homologous polynucleotide sequence” or “homologous amino acidsequence,” or variations thereof, refer to sequences characterized by ahomology at the polynucleotide level or amino acid level. Homologouspolynucleotide sequences encode those sequences coding for isoforms ofCD16. Different genes can encode isoforms such as homologous CD16polynucleotide sequences of species other than mice, including othervertebrates, such as human, frog, rat, rabbit, dog, cat, cow, horse, andother organisms. Homologous polynucleotide sequences also includenaturally occurring allelic variations and mutations of SEQ ID NOs:1 or2. Homologous polynucleotide sequences may encode conservative aminoacid substitutions in SEQ ID NOS:1 or 2.

The invention further encompasses using CD16 polynucleotide moleculesthat differ from the polynucleotide sequence shown in SEQ ID NO:3, dueto degeneracy of the genetic code and thus encode the same CD16 as thatencoded by the polynucleotide sequence shown in SEQ ID NO:3. Anypolynucleotide molecule encoding a polypeptide having an amino acidsequence shown in SEQ ID NOS:1 or 2 is useful for modifying NK-92 cells.

In addition to the CD16 polynucleotide sequence shown in SEQ ID NO:3,DNA sequence polymorphisms that change the CD16 amino acid sequences canalso be useful to modify NK-92 cells. For example, allelic variationsamong individuals exhibit genetic polymorphisms in CD16 genes.

Moreover, CD16 genes from other species that have a polynucleotidesequence that differs from the sequence of SEQ ID NO:3 are contemplatedto be useful in the compositions and methods of the invention.

“CD16 variant polynucleotide” or “CD16 variant polynucleotide sequence”means a polynucleotide molecule which encodes a CD16 polypeptide that(1) has at least about 70% polynucleotide sequence identity with apolynucleotide acid sequence encoding a full-length native CD16, (2) afull-length native CD16 lacking the signal peptide, (3) an extracellulardomain of a CD16, with or without the signal peptide, or (4) any otherfragment of a full-length CD16. Ordinarily, a CD16 variantpolynucleotide will have at least about 70% polynucleotide sequenceidentity, more preferably at least about 71%-99% polynucleotide sequenceidentity and yet more preferably at least about 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% and even more preferably, 99%polynucleotide sequence identity with the polynucleotide sequenceencoding a full-length, native CD16. A CD16 variant polynucleotide canencode full-length native CD16 lacking the signal peptide, anextracellular domain of CD16, with or without the signal sequence, orany other fragment of a full-length CD16.

Ordinarily, CD16 variants are at least about 30 polynucleotides, oftenat least about 60, 90, 120, 150, 180, 210, 240, 270, 300, 450, 600polynucleotides in length, more often at least about 900 polynucleotidesin length, or more.

“Percent (%) polynucleotide sequence identity” with respect toCD16-encoding polynucleotide sequences is defined as the percentage ofpolynucleotides in the CD16 polynucleotide sequence of interest that areidentical with the polynucleotides in a candidate sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment can be achieved invarious ways well-known in the art; for instance, using publiclyavailable software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any necessary algorithms to achievemaximal alignment over the full length of the sequences being compared.

When polynucleotide sequences are aligned, the % polynucleotide sequenceidentity of a given polynucleotide sequence C to, with, or against agiven polynucleotide sequence D (which can alternatively be phrased as agiven polynucleotide sequence C that has or comprises a certain %polynucleotide sequence identity to, with, or against a givenpolynucleotide sequence D) can be calculated as:% polynucleotide sequence identity=W/Z·100where

-   -   W is the number of polynucleotides scored as identical matches        by the sequence alignment program's or algorithm's alignment of        C and D        and    -   Z is the total number of polynucleotides in D.

When the length of polynucleotide sequence C is not equal to the lengthof polynucleotide sequence D, the % polynucleotide sequence identity ofC to D will not equal the % polynucleotide sequence identity of D to C.

Homologs or other related sequences (e.g., paralogs) can be obtained bylow, moderate or high stringency hybridization with all or a portion ofthe particular sequence used as a probe using polynucleotidehybridization and cloning methods well known in the art.

In addition to naturally-occurring allelic variants of CD16polynucleotides, changes can be introduced by mutation into SEQ ID NO:3that incur alterations in the amino acid sequence of CD16 but does notalter CD16 function for the purposes of the invention. For example,polynucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in SEQ ID NOs:1 or 2. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of CD16 without altering CD16 function in themethods and compositions of the invention, whereas an “essential” aminoacid residue is required for activity. For example, amino acid residuesthat are conserved among the CD16 amino acids of the invention areparticularly non-amenable to alteration (Table 9).

Useful conservative substitutions are shown in Table B, “Preferredsubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the subject invention so long as the substitution does notmaterially alter the activity of the compound in the methods andcompositions of the invention. If such substitutions result in such achange, then more substantial changes, indicated in Table C asexemplary, are introduced and the products screened for CD16 activityfor the methods and compositions of the invention.

TABLE B Preferred substitutions Original Exemplary Preferred residuesubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L)Norleucine, Ile, Val, Ile Met, Ala, Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Leu Ala, Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge or (3) hydrophobicity, or (4) the bulk of the side chain ofthe target site can modify CD16 polypeptide function or immunologicalidentity. Residues are divided into groups based on common side-chainproperties as denoted in Table A. Non-conservative substitutions entailexchanging a member of one of these classes for another class.Substitutions may be introduced into conservative substitution sites ormore preferably into non-conserved sites.

TABLE C Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986;Zoller and Smith, 1987), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., 1985) or other known techniques can beperformed on the cloned DNA to produce CD16 variants (Ausubel, 2002;Sambrook and Russell, 2001).

CD16 Polypeptide Variants

“CD16 polypeptide variant” means a CD16 polypeptide having at least: (1)about 80% amino acid sequence identity with a full-length native CD16sequence, (2) a CD16 sequence lacking a signal peptide, (3) anextracellular domain of a CD16, with or without a signal peptide, or (4)any other fragment of a full-length CD16 sequence. For example, CD16variants include those wherein one or more amino acid residues are addedor deleted at the N- or C-terminus of the full-length native amino acidsequence. A CD16 polypeptide variant will have at least about 80% aminoacid sequence identity, preferably at least about 81% amino acidsequence identity, more preferably at least about 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%amino acid sequence identity and most preferably at least about 99%amino acid sequence identity with a full-length native sequence CD16sequence. Ordinarily, CD16 variant polypeptides are at least about 10amino acids in length, often at least about 20 amino acids in length,more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues that are identical with amino acid residues in aCD16 sequence in a candidate sequence when the two sequences arealigned. To determine % amino acid identity, sequences are aligned andif necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Publiclyavailable computer software such as BLAST, BLAST2, ALIGN2 or Megalign(DNASTAR) can be used to align polypeptide sequences. Those skilled inthe art will determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y·100where

-   X is the number of amino acid residues scored as identical matches    by the sequence alignment program's or algorithm's alignment of A    and B    and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A.

A CD16 “chimeric polypeptide” or “fusion polypeptide” comprises CD16fused to a non-CD16 polypeptide. A non-CD16 polypeptide is notsubstantially homologous to CD16 (SEQ ID NOs:1 or 2). A CD16 fusionpolypeptide may include any portion to an entire CD16, including anynumber of biologically active portions.

Fusion polypeptides can be easily created using recombinant methods. Apolynucleotide encoding CD16 (e.g., SEQ ID NO:3) can be fused in-framewith a non-CD16 encoding polynucleotide, to the CD16 N- or C-terminus,or internally. Fusion genes may also be synthesized by conventionaltechniques, including automated DNA synthesizers and PCR amplificationusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (Ausubel, 2002). Manyvectors are commercially available that facilitate sub-cloning CD16in-frame to a fusion moiety.

Measuring Cytoxicity

Some examples of measuring cytoxicity are presented herein below, butany cytotoxicity assay can be used without departing from the spirit andscope of the invention.

NK-92-CD16 cells can be used in ADCC assays as a pure “effector” cellpopulation having defined and consistent characteristics. In someinstances it can be desirable to select and use cells that exhibitintermediate levels of CD16 expression. In particular, such intermediatecells can be of use in generating dose-response curves and as “ladder”type calibrators for CD16 activity. NK-92 cells transduced with the lowand high affinity forms of CD16 could likewise be used for thesepurposes.

Such in-vitro assays are commonly employed for purposes such as thedetermination of the efficacy of antibodies that are being developed aspotential therapeutic agents. ADCC assays are typically performed byloading target cells with an indicator material such as [⁵¹Cr] or aEuropium chelate; treating the indicator-loaded target cells with theantibody to be evaluated; and exposing these cells to NK-92-CD16effector cells. Lysis of the target cells is indicated by the release ofthe indicator material into the assay supernate where its concentrationcan be measured by a suitable method such as scintillation counting(⁵¹Cr) or fluorescence intensity or lifetime determination (Europiumchelate). Efficacy can likewise be assessed by the measurement ofsurrogate indicators such as cytokine release by the NK-92-CD16 cells;the up-regulation of NK cell activation markers, such as CD25, CD69and/or CD95L; activation of NK-92 cell transcription factors, such asNF-AT or NF-κB; or the activation of caspases or other markers ofapoptosis in the target cells. CD16-deficient parental NK-92 cells serveas a unique and valuable control in such assays as they permitdifferentiating between ADCC-mediated cytotoxicity and other cytolyticeffects that NK-92 cells exert on the target cells. The preferred targetcells in ADCC assays are ones that express an antigen that isappropriate to the antibody being evaluated and that have lowsusceptibility to lysis by the parental NK-92 cell line. If such atarget cell line is not conveniently available, other suitable celllines, such as the ovarian carcinoma line SKOV-3 (e.g., ATCC DepositHTB-77) (Tam et al., 1999), can sometimes be used as a viablesubstitute, particularly if transduced/transfected such that theyexpress the specific antigen required. Among the cell lines that havebeen demonstrated to be suitable for use in assays of ADCC-mediatedcytotoxicity are U373MG and T98G (e.g., ATCC Deposit CRL-1690) (Komatsuand Kajiwara, 1998); AML-193 (myeloid; e.g., ATCC Deposit CRL-9589) andSR-91 (lymphoid progenitor) (Gong et al., 1994); and ALL1 and REH(B-cell acute lymphocytic leukemia) (Reid et al., 2002). Other types oftarget cells such as the FcγRII/III⁺ murine mastocytoma cell line P815(e.g., ATCC Deposit No. TIB-64); and the FcγRII/III⁺ myelocytic leukemialine THP-1 (e.g., ATCC Deposit No. TIB-202) that have limited (between 5and 30%) susceptibility to lysis by unmodified NK-92 cells arepreferably used where redirected cytotoxicity or ADCC is to bedetermined in order to ensure sufficient assay dynamic range for thedetection of significant effects through CD16. Other cell types withlimited cytolytic potential that express or are engineered to expressspecific cell surface markers of interest can also be employed astargets. Furthermore, the baseline cytolytic capacity of NK-92 cells canbe reduced by either decreasing the IL-2 concentration in the culturesand/or assaying four days after passing the cells into freshIL-2-containing medium. The functionality of CD16 introduced intoNK-92-CD16, NK-92-CD16-γ or NK-92-CD16-ζ cells can be determined usingthe cytotoxicity assay of Example 4 in either the ADCC or the“redirected cytotoxicity” format. Monoclonal antibodies that bind CD16can be used to test for function of the receptor on NK-92 cells in aredirected cytotoxicity assay, in which the antibody's F(ab) portionbinds the receptor on the NK cell and the Fc portion binds to Fcreceptors on appropriate target cells (FcγRII/III on P815 or THP-1target cells). Another form of redirected cytotoxicity can be testedusing a chimeric bi-specific antibody, such as 2B1 (Clark et al., 1997;Weiner et al., 1995a; Weiner et al., 1995b), which expresses two F(ab)regions, one which binds CD16 on the NK-92 cell and another that bindsthe Her2/neu antigen on an appropriate target cell line, such as SKOV-3.Furthermore, monoclonal antibodies that specifically bind antigens thatare uniquely expressed on the target cells can directly test ADCC. Inthis format, the F(ab) portion of the antibody binds to thecorresponding ligand on the target cell while the CD16 receptor on theNK-92-CD16 cells bind to the Fc portion of the antibody. The resultingcross-link between the antigen on the target cell and the CD16 receptorresults in lysis of the target cell via the ADCC pathway.

For some purposes, particularly as related to the evaluation of bi- orpoly-functional antibodies or in the study of activation mechanisms andother characteristics of NK-92 cells, it can be useful to restructurethe previously described ADCC assay as a “redirected cytotoxicity”assay. For example, a bi-functional antibody having one domain thatspecifically binds to an antigen of interest on the target cells and asecond domain that specifically binds to CD16 on NK-92-CD16 cells can beevaluated in the manner described above. In this instance, thebi-functional antibody cross-links the antigen on the target cell toCD16 on the NK-92-CD16 cell and triggers an ADCC response. The sameassay for research purposes can, for example, treat a target cell thatexpresses CD16 or another Fc receptor with an antibody such as anti-CD16that is directed against this receptor. Exposing the anti-CD16 labeledtarget cells to NK-92-CD16 cells results in the cross-linking of thereceptors on both cells with consequent triggering of ADCC.Differentiation between the ADCC and redirected cytotoxicity formats isbased upon whether the effector cell CD16 receptor binds to the Fcportion of an antibody (ADCC) or this same receptor is bound by theF(ab) portion of an anti-CD16 antibody (redirected cytotoxicity). Asboth binding arrangements can trigger similar cytotoxicity responses inthe target cell, the choice between the ADCC and redirected formats islargely a matter of convenience.

In some instances, it can be advantageous to block known activatingreceptors on NK-92 cells; such methods and agents are well-known; seefor example (Pende et al., 1999; Pessino et al., 1998; Vitale et al.,1998). For example, masking antibodies can be used (Pessino et al.,1998).

In the presence of an appropriate antibody, NK-92-CD16 cells caneffectively and efficiently lyse target cells. CD16 in NK cells isnon-covalently associated in the plasma membrane with homodimers orheterodimers of the FcεRI-γ (Genbank Accession No. M33195; SEQ ID NO:4(polynucleotide) and SEQ ID NO:5 (polypeptide)) or TCR-ζ (GenbankAccession No. J04132; SEQ ID NO:6 (polynucleotide); SEQ ID NO:7(polypeptide)) accessory signaling proteins; these sequences arepresented in Tables 4-7. Discussion regarding sequence identity appliesalso to SEQ ID NOs:4-7; thus, those polynucleotides or polypeptides ofSEQ ID NOs:4-7 having about at least 70%-100% sequence identity, as wellas 80%-90%, and 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity are usefulfor the methods and composition of the invention. When stimulated by thebinding of Fc to CD16, these accessory signaling proteins can transduceintracellular signals that activate NK cell cytotoxicity and cytokinerelease activities. In addition, they support surface expression ofCD16. The signaling activity initiated through ζ and γ serves to triggerADCC responses. FcεRI-γ and/or TCR-ζ can conveniently be co-introducedinto NK-92 cells by sequential transduction with CD16 and FcεRI-γ and/orTCR-ζ cDNA in the manner described above. In this process, it isgenerally desirable to delete the EGFP gene from the vector containingthe CD16 cDNA. The sequential transduction process consists oftransducing the parental NK-92 cells with the CD16 vector;immunostaining the transduced cells with a fluorescently labeledanti-CD16 antibody; sorting the cells for CD16 expression; transducingthe NK-92-CD16 cells with a vector containing cDNA for both theaccessory protein and EGFP; and sorting the doubly transduced cells onthe basis of EGFP expression. The resulting doubly transduced NK-92cells (NK-92-CD16/γ or NK-92-CD16/ζ, respectively) usually exhibithigher levels of surface CD16 expression and enhanced cytotoxicity andcytokine release activities than do NK-92-CD16 cells.

TABLE 4 FcεRI-γ polynucleotide sequence (SEQ ID NO: 4) cagaacggccgatctccagc ccaagatgat tccagcagtg gtcttgctct tactcctttt 60 ggttgaacaagcagcggccc tgggagagcc tcagctctgc tatatcctgg atgccatcct 120 gtttctgtatggaattgtcc tcaccctcct ctactgtcga ctgaagatcc aagtgcgaaa 180 ggcagctataaccagctatg agaaatcaga tggtgtttac acgggcctga gcaccaggaa 240 ccaggagacttacgagactc tgaagcatga gaaaccacca cagtagcttt agaatagatg 300 cggtcatattcttctttggc ttctggttct tccagccctc atggttggca tcacatatgc 360 ctgcatgccattaacaccag ctggccctac ccctataatg atcctgtgtc ctaaattaat 420 atacaccagtggttcctcct ccctgttaaa gactaatgct cagatgctgt ttacggatat 480 ttatattctagtctcactct cttgtcccac ccttcttctc ttccccattc ccaactccag 540 ctaaaatatgggaagggaga acccccaata aaactgccat ggactggact c 591

TABLE 5 FcεRI-γ polypeptide sequence (SEQ ID NO: 5) Met Ile Pro Ala ValVal Leu Leu Leu Leu Leu Leu Val Glu Gln Ala1               5                   10                  15 Ala Ala LeuGly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu            20                  25                  30 Phe Leu Tyr GlyIle Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile        35                  40                  45 Gln Val Arg Lys AlaAla Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val    50                  55                  60 Tyr Thr Gly Leu Ser ThrArg Asn Gln Glu Thr Tyr Glu Thr Leu Lys65                  70                  75                  80 His GluLys Pro Pro Gln                 85

TABLE 6 TCR-ζ polynucleotide sequence (SEQ ID NO: 6) cttttctcctaaccgtcccg gccaccgctg cctcagcctc tgcctcccag cctctttctg 60 agggaaaggacaagatgaag tggaaggcgc ttttcaccgc ggccatcctg caggcacagt 120 tgccgattacagaggcacag agctttggcc tgctggatcc caaactctgc tacctgctgg 180 atggaatcctcttcatctat ggtgtcattc tcactgcctt gttcctgaga gtgaagttca 240 gcaggagcgcagagcccccc gcgtaccagc agggccagaa ccagctctat aacgagctca 300 atctaggacgaagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga 360 tggggggaaagccgagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 420 ataagatggcggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg 480 ggcacgatggcctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc 540 acatgcaggccctgccccct cgctaacagc caggggattt caccactcaa aggccagacc 600 tgcagacgcccagattatga gacacaggat gaagcattta caacccggtt cactcttctc 660 agccactgaagtattcccct ttatgtacag gatgctttgg ttatatttag ctccaaacct 720 tcacacacagactgttgtcc ctgcactctt taagggagtg tactcccagg gcttacggcc 780 ctgccttgggccctctggtt tgccggtggt gcaggtagac ctgtctcctg gcggttcctc 840 gttctccctgggaggcgggc gcactgcctc tcacagctga gttgttgagt ctgttttgta 900 aagtccccagagaaagcgca gatgctagca catgccctaa tgtctgtatc actctgtgtc 960 tgagtggcttcactcctgct gtaaatttgg cttctgttgt caccttcacc tcctttcaag 1020 gtaactgtactgggccatgt tgtgcctccc tggtgagagg gccgggcaga ggggcagatg 1080 gaaaggagcctaggccaggt gcaaccaggg agctgcaggg gcatgggaag gtgggcgggc 1140 aggggagggtcagccagggc ctgcgagggc agcgggagcc tccctgcctc aggcctctgt 1200 gccgcaccattgaactgtac catgtgctac aggggccaga agatgaacag actgaccttg 1260 atgagctgtgcacaaagtgg cataaaaaac agtgtggtta cacagtgtga ataaagtgct 1320 gcggagcaagaggaggccgt tgattcactt cacgctttca gcgaatgaca aaatcatctt 1380 tgtgaaggcctcgcaggaag acgcaacaca tgggacctat aactgcccag cggacagtgg 1440 caggacaggaaaaacccgtc aatgtactag gg 1472

TABLE 7 TCR-ζ polypeptide sequence (SEQ ID NO: 7) Met Lys Trp Lys AlaLeu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu1               5                   10                  15 Pro Ile ThrGlu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys            20                  25                  30 Tyr Leu Leu AspGly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala        35                  40                  45 Leu Phe Leu Arg ValLys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr    50                  55                  60 Gln Gln Gly Gln Asn GlnLeu Tyr Asn Glu Leu Asn Leu Gly Arg Arg65                  70                  75                  80 Glu GluTyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met                85                  90                  95 Gly Gly LysPro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu            100                 105                 110 Leu Gln Lys AspLys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys        115                 120                 125 Gly Glu Arg Arg ArgGly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu    130                 135                 140 Ser Thr Ala Thr Lys AspThr Tyr Asp Ala Leu His Met Gln Ala Leu145                 150                 155                 160 Pro ProArg

NK-92 cells are widely used as a model system for the study of NK cellactivation, action and inhibition as they stably express a defined suiteof known NK cell activating receptors including NKp46, NKp44, 2B4, andNKG2D, but lack CD16, activating KIR, and NKG2C. More importantly, theyalso lack almost all of the known NK cell inhibitory receptors exceptlow levels of NKG2A/CD94 and ILT2/LIR1. This is a major advantage of thepresent invention since MHC class I molecules, which are expressed onmost cells and serve as ligands for NK cell inhibitory receptors, do noteffectively inhibit the activation of NK-92 cells. Furthermore, thepresent invention restores the CD16 activating receptor to NK-92 cellsthereby increasing the range of activating ligands to which these cellsrespond. Both the activating receptors and any residual inhibitoryreceptors can be selectively blocked by the treatment of the effectorcells with the appropriate monoclonal antibodies or the correspondingF_((ab′)2) fragments, such as 3.43.13 (anti-NKp44), 9E2 (anti-NKp46),158 (anti-2B4), and 3G8 (anti-CD16). The ability to selectively blockindividual receptors or groups of receptors in conjunction with targetcells that differ in susceptibility to lysis by NK-92 cells (andderivatives thereof) facilitates the study of the many mechanismsinvolved in NK cell activation and inhibition.

Antibodies (Abs) The invention makes use of Abs and antibody fragments,such as F_(ab) or (F_(ab′))₂, that bind immunospecifically to theirepitopes.

“Antibody” (Ab) comprises single Abs directed against an epitope,anti-Ab compositions with poly-epitope specificity, single chainanti-Abs, and fragments of Abs. A “monoclonal antibody” is obtained froma population of substantially homogeneous Abs, i.e., the individual Abscomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Exemplary Abs include polyclonal (pAb), monoclonal (mAb), humanized,bi-specific (bsAb), chimeric and heteroconjugate Abs. Antibodies can beproduced by any known method in the art or obtained commercially.

Monovalent Abs

The Abs may be monovalent Abs that consequently do not cross-link witheach other. For example, one method involves recombinant expression ofimmunoglobulin (Ig) light chain and modified heavy chains. Heavy chaintruncations generally at any point in the F_(c) region will preventheavy chain cross-linking. Alternatively, the relevant cysteine residuesare substituted with another amino acid residue or are deleted,preventing cross-linking. In-vitro methods are also suitable forpreparing monovalent Abs. Abs can be digested to produce fragments, suchas F_(ab) fragments (Harlow and Lane, 1988; Harlow and Lane, 1999).

Humanized and Human Abs

Abs may further comprise humanized or human Abs. Humanized forms ofnon-human Abs are chimeric Igs, Ig chains or fragments (such as F_(v),F_(ab), F_(ab′), F_((ab′)2) or other antigen-binding subsequences ofAbs) that contain minimal sequence derived from non-human Ig.

Generally, a humanized antibody has one or more amino acid residuesintroduced from a non-human source. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization is accomplished bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody (Jones et al., 1986; Riechmann et al.,1988; Verhoeyen et al., 1988). Such “humanized” Abs are chimeric Abs(1989), wherein substantially less than an intact human variable domainhas been substituted by the corresponding sequence from a non-humanspecies. In practice, humanized Abs are typically human Abs in whichsome CDR residues and possibly some Fc residues are substituted byresidues from analogous sites in rodent Abs. Humanized Abs include humanIgs (recipient antibody) in which residues from a complementarydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit, having the desired specificity, affinity and capacity. In someinstances, corresponding non-human residues replace F_(v) frameworkresidues of the human Ig. Humanized Abs may comprise residues that arefound neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody comprisessubstantially all of at least one, and typically two, variable domains,in which most if not all of the CDR regions correspond to those of anon-human Ig and most if not all of the FR regions are those of a humanIg consensus sequence. The humanized antibody optimally also comprisesat least a portion of an Ig constant region (F_(c)), typically that of ahuman Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

Human Abs can also be produced using various techniques, including phagedisplay libraries (Hoogenboom et al., 1991; Marks et al., 1991) and thepreparation of human mAbs (Boerner et al., 1991; Reisfeld and Sell,1985). Similarly, introducing human Ig genes into transgenic animals inwhich the endogenous Ig genes have been partially or completelyinactivated can be exploited to synthesize human Abs. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire (1997a; 1997b; 1997c; 1997d; 1997; 1997; Fishwild etal., 1996; 1997; 1997; 2001; 1996; 1997; 1997; 1997; Lonberg and Huszar,1995; Lonberg et al., 1994; Marks et al., 1992; 1997; 1997; 1997).

Bi-Specific mAbs

Bi-specific Abs are monoclonal, preferably human or humanized, that havebinding specificities for at least two different antigens.

Traditionally, the recombinant production of bi-specific Abs is based onthe co-expression of two Ig heavy-chain/light-chain pairs, where the twoheavy chains have different specificities (Milstein and Cuello, 1983).Because of the random assortment of Ig heavy and light chains, theresulting hybridomas (quadromas) produce a potential mixture of tendifferent antibody molecules, of which only one has the desiredbi-specific structure. The desired antibody can be purified usingaffinity chromatography or other techniques (Traunecker et al., 1991;1993).

To manufacture a bi-specific antibody (Suresh et al., 1986), variabledomains with the desired antibody-antigen combining sites are fused toIg constant domain sequences. The fusion is preferably with an Igheavy-chain constant domain, comprising at least part of the hinge, CH2,and CH3 regions. Preferably, the first heavy-chain constant region (CH1)containing the site necessary for light-chain binding is in at least oneof the fusions. DNAs encoding the Ig heavy-chain fusions and, ifdesired, the Ig light chain, are inserted into separate expressionvectors and are co-transfected into a suitable host organism.

The interface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture (1996). The preferred interface comprises atleast part of the CH3 region of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This mechanism increases theyield of the heterodimer over unwanted end products such as homodimers.

Bi-specific Abs can be prepared as full length Abs or antibody fragments(e.g. F_((ab′)2) bi-specific Abs). One technique to generate bi-specificAbs exploits chemical linkage. Intact Abs can be proteolytically cleavedto generate F_((ab′)2) fragments (Brennan et al., 1985). Fragments arereduced with a dithiol complexing agent, such as sodium arsenite, tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The generated F_(ab′) fragments are then converted tothionitrobenzoate (TNB) derivatives. One of the F_(ab′)-TNB derivativesis then reconverted to the F_(ab′)-thiol by reduction withmercaptoethylamine and is mixed with an equimolar amount of the otherF_(ab′)-TNB derivative to form the bi-specific antibody. The producedbi-specific Abs can be used as agents for the selective immobilizationof enzymes.

F_(ab′) fragments may be directly recovered from E. coli and chemicallycoupled to form bi-specific Abs. For example, fully humanizedbi-specific F_((ab′)2) Abs can be produced (Shalaby et al., 1992). EachF_(ab′) fragment is separately secreted from E. coli and directlycoupled chemically in-vitro, forming the bi-specific antibody.

Various techniques for making and isolating bi-specific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, leucine zipper motifs can be exploited (Kostelnyet al., 1992). Peptides from the Fos and Jun proteins are linked to theF_(ab′) portions of two different Abs by gene fusion. The antibodyhomodimers are reduced at the hinge region to form monomers and thenre-oxidized to form antibody heterodimers. This method can also produceantibody homodimers. The “diabody” technology (Holliger et al., 1993)provides an alternative method to generate bi-specific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker that istoo short to allow pairing between the two domains on the same chain.The V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, forming twoantigen-binding sites. Another strategy for making bi-specific antibodyfragments is the use of single-chain F_(v) (sF_(v)) dimers (Gruber etal., 1994). Abs with more than two valencies are also contemplated, suchas tri-specific Abs (Tutt et al., 1991).

Heteroconjugate Abs

Heteroconjugate Abs, consisting of two covalently joined Abs, have beenproposed to target immune system cells to unwanted cells (1987) and fortreatment of human immunodeficiency virus (HIV) infection (1991; 1992).Abs prepared in-vitro using synthetic protein chemistry methods,including those involving cross-linking agents, are contemplated. Forexample, immunotoxins may be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsinclude iminothiolate and methyl-4-mercaptobutyrimidate (1987).

Pharmaceutical Compositions for Abs

Abs can be administered in pharmaceutical compositions. Principles andconsiderations involved in preparing such compositions, as well asguidance in the choice of components, can be found in (de Boer, 1994;Gennaro, 2000; Lee, 1990).

Liposomes can also be used as a delivery vehicle for intracellularintroduction. Where antibody fragments are used, the smallest inhibitoryfragment that specifically binds to the epitope is preferred. Forexample, peptide molecules can be designed that bind a preferred epitopebased on the variable-region sequences of a useful antibody. Suchpeptides can be synthesized chemically and/or produced by recombinantDNA technology (Marasco et al., 1993). Formulations may also containmore than one active compound for a particular treatment, preferablythose with activities that do not adversely affect each other. Thecomposition can comprise an agent that enhances function, such as acytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitoryagent. The composition can also contain cells, such as NK-92 cells.

The active ingredients can also be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization; forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules) or in macro emulsions.

The formulations to be used for in vivo administration are highlypreferred to be sterile. This is readily accomplished by filtrationthrough sterile filtration membranes or any of a number of techniques.

Sustained-release preparations may also be prepared, such assemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (Boswell and Scribner, 1973),copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as injectable microspheres composed of lactic acid-glycolic acidcopolymer, and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods and may be preferred.

In a similar manner, the use of multiple antibodies or F_((ab′)2)fragments to specifically block selected activating receptors, permitsrefinement of the identification of the receptors that are involved inany particular case. In this regard, the presence of NK cell-mediatedlysis of a target for which all known activating receptors are blockedis suggestive of the presence of previously unknown activating receptorsor ligands. The converse experiment of observing the cytolytic activityof native and transduced NK-92 cells against target cells that have beenmodified so as to express putative ligands for these receptors canprovide additional information and possible confirmation. The cytokineresponse of the effector cells in these experiments can also provideadditional information. Therefore, the invention offers a myriad ofoptions for refining the assay conditions to suit the user's needs, aswell as providing a valuable tool for investigations of basicphysiological mechanisms of NK cell function.

In addition, the use of antibodies for the therapeutic treatment ofdisease is a rapidly growing and evolving field. Although the mechanismsinvolved in the therapeutic effects of antibody treatments are stillbeing elucidated, there is evidence that these effects are mediated bythe ad-hoc interaction of the Fc portion of the administered antibodywith the corresponding Fc receptors on cytolytic effector cells such asneutrophils, mononuclear phagocytes, transformed cells, T-cells and NKcells. The cytolytic activity of the effector cells is thereby directedagainst those target cells that display surface antigens that are boundby the antibody. This proposed mechanism is not meant to limit theinvention in any way.

The major current thrust of this field involves the use of full-lengthhumanized monoclonal antibodies (thus containing Fc domains) that aredirected toward cell surface antigens on tumor cells. For example,substantial clinical benefits have been demonstrated for the treatmentof certain breast cancers with the anti-Her2/neu antibody, Herceptin,and the treatment of B cell leukemias with the anti-CD20 antibody,Rituximab. CD16⁺ effector cells such as native NK cells and theNK-92-CD16 cells and NK-92-CD16γ/ζ cells of this invention bind to theFc portions of Herceptin or Rituximab antibodies that, in turn, bind viatheir F_((ab)) portions to the corresponding cell surface antigen on acancer cell. This ligation of the cancer cell antigen to CD16 activatesthe effector cell and directs its cytolytic activity against the cancercell, thus resulting in its destruction.

Another aspect of development in this area is directed toward thecreation of chimeric antibodies that incorporate two or moreantigen-binding [F_((ab))] domains having differing specificities. Achimeric “bi-specific antibody” can, by way of example, incorporate oneF_((ab)) binding domain that specifically binds to a cell surface markerthat is uniquely or characteristically expressed on the target tumor orinfected cells and a second F_((ab)) domain that specifically engagesactivating receptors such as CD16 on NK or other effector cells. Suchchimeric antibodies are exemplified by the monoclonal antibody 2B1(Clark et al., 1997; Weiner et al., 1995a; Weiner et al., 1995b) whichincorporates one F_((ab)) domain that specifically binds to the ErbB2(HER2/neu) antigen and a second F_((ab)) domain that specifically bindsto CD16. Cells of the ErbB2⁺ ovarian cancer line SKOV-3 are onlyslightly susceptible to cytolysis by NK cells or NK-92-CD16 cells.However, SKOV-3 cells become highly susceptible to NK-92-CD16cell-mediated cytolysis in the presence of 2B1 antibody which ligatesthe ErbB2 antigen on a SKOV-3 cell to the CD16 activating receptor on aNK-92-CD16 cell. (FIG. 5).

Groner and Moritz (Groner and Moritz, 1997) describe yet another meansof using antibodies to direct the activity of an effector cell against aspecific target. In this approach, the effector cell is geneticallyengineered to express a single polypeptide chain consisting of anantigen-specific monovalent F_((ab)) binding domain that is covalentlylinked to a signaling domain, such as TCR-ζ (Genbank Accession No.J04132; SEQ ID NO:6).

As illustrated in greater detail below in Example 6, ADCC and redirectedcytotoxicity assays can be used to identify antibodies and antibodyconstructs that are useful as therapeutic agents for the treatment ofcancers and infections. NK-92-CD16, NK-92-CD16-γ or NK-92-CD16-ζ cellsare used as effector cells in these assays. The target cells and thecharacteristics of the antibody or antibody construct to be evaluatedlargely determine whether the ADCC or the redirected cytotoxicity assayformat is more appropriate. A target cell that expresses the ligand ofinterest, but which does not express any Fc receptors is appropriate forthe evaluation of antibodies in the ADCC format. This same type oftarget cell is also suitable for the ADCC evaluation of antibodyconstructs that include an Fc domain. Conversely, an antibody constructthat incorporates separate F_((ab)) binding domains that are specificfor the antigen of interest and for CD16 are most appropriatelyevaluated via redirected cytotoxicity using target cells that expressthe antigen of interest, but which do not express CD16. Other sucharrangements are likewise possible and can be used in the practice ofthe present invention. In order to ensure maximum assay dynamic range,it is desirable to select the target cell from among those that areminimally susceptible to lysis by the parental NK-92 cell line. For thisreason, the target cell is typically selected to exhibit NK-92 mediatedlysis of between 0% and 30%, preferably between 0% and 20%, morepreferably between 0% and 10% and most preferably between 0% and 5%.Target cell lines such as SKOV-3 are useful because they exhibit minimal(5%-30%) susceptibility to lysis by NK-92 cells and they constitutivelyexpress certain cell surface antigens that are of particular interest astargets for therapeutic antibodies. These cells can also be transducedor transfected to express other antigens of interest and utility.

One significant application of the present invention is in the screeningof hybridoma supernates for the presence of ADCC-inducing monoclonalantibodies. The spleen cell fusions employed for the initial generationof monoclonal antibodies result in a heterogeneous population of cells,some of which produce antibodies. Each individual antibody-producingcell in this mixture typically produces a unique antibody that has atleast some affinity for the target antigen. The cells in this originalheterogeneous mixture are sub-cloned, typically by limiting dilution, tothe point where each sub-clone originates from a single parent cell.Each sub-clone is then typically screened to eliminate those that do notsecrete immunoglobulins. The remaining sub-clones, which can number inthe tens to hundreds, each secrete a unique antibody, a few of which canhave specificities, affinities and other characteristics that make themsuitable for further evaluation and development as potential therapeuticagents. The ADCC and redirected cytotoxicity assays of Example 6 findutility in the screening of clones to identify those sub-clones thatsecrete potentially useful antibodies of the IgG isotypes. These sameassays can subsequently be used to support the evaluation,characterization and further development of these antibodies.

The present invention consists of NK-92 cell constructs that have beenengineered to stably express the fully active, high or low affinity formof the FcγRIII (CD16) receptor. CD16 is naturally present on NK cells,but is not expressed on parental NK-92 cells and or with any stabilityby any of the other known NK-like cell lines. The transduced NK-92-CD16,NK-92-CD16-γ and NK-92-CD16-ζ cell lines are, therefore, unique amongthe presently available NK-like cell lines. Furthermore, NK-92 cells andtheir genetically engineered derivatives exhibit levels of functionalresponsiveness in cytotoxicity and cytokine assays that are superior toprimary NK cells and most of the other available human NK-like celllines and appear to be safely tolerated in human subjects. For thesereasons, the constructs of the present invention, when used incombination with an antibody that specifically binds to a cell surfacemarker that is uniquely or characteristically expressed on the intendedtarget cell, provides a substantially higher level of cytolytic activityand specificity toward the target cell type than is provided by parentalNK-92 cells or the antibody alone when used in the same manner.Furthermore, unlike native NK cells, the NK-92 cell constructs of thisinvention express minimal inhibitory receptors, which makes themreactive toward a broad array of tumors. The target cell specificity ofthese constructs is determined by the co-administered antibody orantibodies, thus permitting these constructs to be used without changefor any clinical indication for which a suitable antibody can beprepared. Polyclonal antibodies, cocktails consisting of multiplemonoclonal antibodies, and chimeric antibody constructs such asbi-specific antibodies, mini-bodies and TriBi (trimeric, bi-specific)antibodies can be beneficially used in conjunction with this invention.

The present invention can be administered either to animals or to humansubjects. When evaluating clinical efficacy, the most beneficial animalmodels include RAG-deficient/common γ chain-deficient mice (lacking T,B, and NK cells) or SCID mice (lacking T and B cells); these strains areavailable from The Jackson Laboratory; Bar Harbor, Me. The suppressionof the native immune system in such immuno-compromised animalsfacilitates differentiation between responses induced by the treatmentand normal immune responses. Furthermore, the cell line derivatives canbe used to treat animal subjects, such as bovines, swine, rabbits,alpacas, horses, canines, felines, ferrets, rats, mice, fowl andbuffalo, suffering from tumors in combination with tumor-specificantibodies. The therapeutic use of NK-92 cells can be enhanced bypreparing subjects who are to receive CD16-transduced NK-92 cells fortherapy by infusion the subjects with a low dose of IL-2 for severaldays prior to administering the NK-92-CD16 cells and targeting antibody,and to continue this infusion for several days afterwards.Alternatively, the CD16-transduced NK-92 cells can be prepared fromcells of the NK-92 ml or NK-92ci cell lines that have been engineered toconstitutively express IL-2. In either case, concurrent treatment withIL-2 can increase the survival of the administered NK-92 cells.

Since the NK-92 cell line was isolated from a large granular lymphomasubject, the cells have the potential to establish tumors in recipientsubjects. Although this tumorigenicity has not been observed in anysubjects (human or animal), accepted practice incorporates γ irradiatingNK-92 cells prior to administration at doses levels that suppressesNK-92 cell proliferation while substantially maintaining cytotoxicityand cell survival. Gamma irradiation of NK-92 cells at doses of betweenabout 750 and 1000 Grays, e.g., 750, 800, 850, 900 and 950 Grays, isconsidered to be sufficient for this purpose.

In-vivo treatment of a subject is initiated by administration of thetargeting antibody prior to, or concurrently with, the administration ofCD16-transduced NK-92 cells. Administration is typically via intravenousor intraperitoneal infusion although direct injection into solid tumorsor other such focal lesions can also be used. A split-dose regimen canbe preferable, particularly when IL-2 is not being co-administered, inorder to maintain a high level of active, transduced NK-92 cells in thesubject. In some cases, administering the antibody by infusion and thetransduced cells by direct injection can be advantageous. The efficacyof the treatment is generally assessed by lesion reduction/clearance,cytokine profile or other physiological parameters.

The NK-92-CD16, NK-92-CD16/γ and NK-92-CD16/ζ cells of the presentinvention represent stable and reproducible populations of effectorcells that are of particular utility in the in-vitro evaluation ofantibodies that are being developed as potential therapeutic agents.These same cell lines are effective components of in-vivo therapies forthe treatment of diseases, including cancers and infections.Co-administration of one of these cell lines in conjunction with anantibody that specifically binds to an antigen that is expressed by thetumor or infected cell can potentiate the therapeutic effects of theantibody, thus treating the disease. A similar beneficial effect can beobserved in such cases where the cell line is administered as a soletherapy to a subject that has developed endogenous antibodies against atumor or infection.

In addition, while the Fc receptor CD16 has been exemplified, thepresent invention is not limited to the expression of FCγRIII-A, and theexpression of other Fc receptors for IgG and other antibody types isalso within the scope of the invention, including the classes of Fcγreceptors (e.g., FCγRI (CD64), FCγRII (CD32), FCγRIII, and FcRn, Fcα(alpha), Fcε (epsilon), and their various subclasses. Additional NK-92variant cell lines also fall within the spirit and scope of thisinvention. NK-92 can, by way of example, be co-transduced with genesleading to the creation of cell lines such as NK-92ci-CD16 and NK-92ml-CD16 that express both IL-2 and CD16, and that therefore can be usedwithout need for exogenous IL-2. Other benefits and uses of the presentinvention will be made apparent in the specific examples describedbelow.

Pharmaceutical Compositions

Cells (e.g., modified and unmodified NK-92 cells), polypeptides, andAbs, and derivatives, fragments, analogs and homologs thereof, can beincorporated into pharmaceutical compositions. Such compositionstypically comprise the cell, polypeptide, and/or antibody and apharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration(Gennaro, 2000). Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. Supplementary activecompounds can also be incorporated into the compositions.

Injectable Formulations

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an NK-92 cell and/or antibody) in the required amount inan appropriate solvent with one or a combination of ingredients asrequired, followed by sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium, and the other required ingredientsas discussed. Sterile powders are used for the preparation of sterileinjectable solutions. Methods of preparation include vacuum drying andfreeze-drying that yield a powder containing the active ingredient andany desired ingredient from sterile solutions.

EXAMPLES

The following example is for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

Example 1 CD16 Recombinant Retrovirus Preparation

CD16 cDNA X52645.1 encoding the low affinity form of the transmembraneimmunoglobulin γ Fc region receptor III-A (FcγRIII-A or CD16)[Phenylalanine-157 (F157), complete sequence: SwissProt P08637 (SEQ IDNO:1)] or a polymorphic variant encoding a higher affinity form of theCD16 receptor [Valine-157 (F157V), complete sequence: SwissProtVAR_(—)008801 (SEQ ID NO:2)] was sub-cloned into the bi-cistronicretroviral expression vector, pBMN-IRES-EGFP (obtained from G. Nolan,Stanford University, Stanford, Calif.) using the BamHI and NotIrestriction sites in accordance with standard methods.

The recombinant vector was mixed with 10 μL of PLUS™ Reagent(Invitrogen; Carlsbad, Calif.); diluted to 100 μL with pre-warmed,serum-free Opti-MEM® (Invitrogen; MEM, minimum essential media); furtherdiluted by the addition of 8 μL Lipofectamine™ (Invitrogen) in 100 μLpre-warmed serum-free Opti-MEM®; and incubated at room temperature for15 minutes. This mixture was then brought to a total volume of 1 mL bythe addition of pre-warmed serum-free Opti-MEM®. Phoenix-Amphotropicpackaging cells (obtained from G. Nolan, Stanford University, Stanford,Calif.; (Kinsella and Nolan, 1996)) were grown to 70-80% confluence in a6-well plate and washed with 6 mL of pre-warmed serum-free Opti-MEM®medium (Invitrogen). After removal of the medium, 1 mL of the solutionof recombinant vector in Lipofectamine™ PLUS™ Reagent was added to eachwell, and the cells were incubated for at least three hours at 37° C.under a 7% CO₂/balance air atmosphere. Four mL of pre-warmed RPMI mediumcontaining 10% fetal bovine serum (FBS) was added to each well, and thecells incubated overnight at 37° C., under a 7% CO₂/balance airatmosphere. The media was then removed; the cells washed with 6 mLpre-warmed serum-free Opti-MEM®; 2 mL serum-free Opti-MEM® added; andthe cells incubated at 37° C., under a 7% CO₂/balance air atmosphere foran additional 48 hours.

The virus-containing supernate was collected into a 15 mL plasticcentrifuge tube; centrifuged at 1300 rpm for 5 minutes to remove cellsand cell fragments; and the supernate transferred to another 15 mLplastic centrifuge tube. Immediately before use, 20 μL of PLUS™ Reagentwas added to the virus suspension; the mixture incubated at roomtemperature for 15 minutes; 8 μL Lipofectamine™ added to the mixture;and the mixture incubated for an additional 15 minutes at roomtemperature.

Example 2 Retroviral Transduction of CD16 into NK-92 Cells

NK-92 cells cultured in A-MEM (Sigma; St. Louis, Mo.) supplemented with12.5% FBS, 12.5% fetal horse serum (FHS) and 500 IU rhIL-2/mL (Chiron;Emeryville, Calif.) were collected by centrifugation at 1300 rpm for 5minutes, and the cell pellet was re-suspended in 10 mL serum-freeOpti-MEM® medium. An aliquot of cell suspension containing 5×10⁴ cellswas sedimented at 1300 rpm for 5 minutes; the cell pellet re-suspendedin 2 mL of the retrovirus suspension described in Example 1, and thecells plated into 12-well culture plates. The plates were centrifuged at1800 rpm for 30 minutes and incubated at 37° C. under an atmosphere of7% CO₂/balance air for 3 hours. This cycle of centrifugation andincubation was then repeated a second time. The cells were diluted with8 mL of α-MEM, transferred to a T-25 flask, and incubated at 37° C.under a 7% CO₂/balance air until the cells were confluent. Thetransduced cells were collected, re-suspended in serum-free Opti-MEM®medium, and sorted on the basis of their level of EGFP expression usinga fluorescence activated cell sorter (FACS), EGFP being co-expressedwith, and a surrogate marker for, CD16. Cell-surface expression of CD16was confirmed by immuno-staining the transduced cells with an anti-CD16antibody. The transduced cells, which are designated as NK-92-CD16, werepassed with fresh IL-2 every 4 days and assayed for cell-surfaceexpression of CD16 before use.

FIGS. 1A and 1B (FIG. 1) are flow cytometer scatter diagrams showingNK-92 cells transduced with CD16 cDNA using the pBMN-IRES-EGFP vectorafter staining with secondary phycoerythrin (PE)-conjugated anti-mouseIgG antibody alone (FIG. 1A) or anti-CD 16 antibody (3G8 (Fleit et al.,1982; Perussia and Trinchieri, 1984); mouse IgG)+PE-anti-mouse IgG (FIG.1B) and analysis using a FACS (Becton Dickinson; Franklin Lakes, N.J.)flow cytometer. EGFP expression is assessed on the x-axis and surfaceCD16 expression is on the y-axis. FIG. 1 illustrates that the NK-92-CD16cell line expresses CD16 on the cell surface when stained with amonoclonal anti-CD16 antibody.

Example 3 NK-92-CD16 Cells Co-Expressing CD16 and an Accessory SignalingProtein FcεRI-γ or TCR-ζ

Recombinant retroviruses incorporating inserted genes for the expressionof either accessory signaling proteins, FcεRI-γ (SEQ ID NO:5) or TCR-ζ(SEQ ID NO:7), were prepared by using standard methods to ligate thecorresponding cDNA into the pBMN-IRES-EGFP vector and transfecting thisconstruct into the Phoenix-Amphotropic packaging cell line in thepresence of Lipofectamine™ Plus as described in Example 1. The resultingγ or ζ recombinant retroviruses were used to transduce NK-92 cells asdescribed in Example 2 with the following further modifications.

NK-92 cells transduced with FcεRI-γ polynucleotide (SEQ ID NO:4) orTCR-ζ (SEQ ID NO:6) were collected, re-suspended in serum-free Opti-MEM®medium and sorted on the basis of their level of the co-expressed EGFPusing a FACS, CD16 cDNA was ligated into a version of the pBMN vectorlacking the IRES and EGFP sequences, called pBMN-NoGFP (Yusa et al.,2002). The γ- or ζ-transduced NK-92 cells were secondarily co-transducedwith CD16-pBMN-NoGFP using the same retroviral transduction method asdescribed in Example 2. The co-transduced cells were suspended in α-MEM,transferred to a T-25 flask, and grown to confluency at 37° C. under a7% CO₂/balance air. After reaching confluency, the co-transduced cellswere immuno-stained with an anti-CD16 antibody and sorted by FACS forcell-surface expression of CD16. The selected cells were sub-culturedwith fresh IL-2 every four days and assayed for cell-surface expressionof CD16 before use.

In this Example, only one of the two vectors contained the gene forEGFP. This arrangement facilitates the determination of the levels atwhich both the accessory signaling protein and CD16 are expressed. Inthis case, the accessory signaling protein was co-expressed with EGFP.Thus EGFP fluorescence is a surrogate indicator for the level ofexpression of the accessory signaling protein. An anti-CD16 antibodyconjugated to a fluorophore having an emission spectrum different fromthat of EGFP was employed to determine the level of expression of CD16.

FIG. 2 shows flow cytometer scatter diagrams showing the expression ofCD16 by NK-92 cells transduced with CD16 alone (FIG. 2A) and theincrease in CD16 expression when NK-92 cells are transduced with CD16cDNA in combination with FcεRI-γ cDNA (γ; FIG. 2B); or CD3 ζ cDNA (ζ,FIG. 2C). FIG. 2 shows that when CD16 is co-expressed with FcεRI-γ orCD3 ζ in the NK-92 cell line, the cell-surface expression of CD16 isincreased over that obtained when NK-92 cells are transduced with CD16alone.

Example 4 Cytotoxicity Assays

Effector cells (NK-92, NK-92-CD16, NK-92-CD16ζ, NK-92-CD16γ) were washedby suspension in α-MEM (without IL-2) and sedimented at 1300 rpm for 5minutes. The cell pellet was suspended in α-MEM, cells counted, andaliquots prepared at cell concentrations of 1×10⁵/mL (effector to targetcell ratio (E:T)=1:1), 5×10⁵/mL (E:T=5:1), 1×10⁶/mL (E:T=10:1), 2×10⁶/mL(E:T=20:1) or as appropriate to the determination being performed. Thetransduced NK-92 cells used in these assays were generally selected formaximal CD16 expression as previously described.

The type of target cell used in these assays was selected on the basisof the requirements of the particular determination being performed.Raji cells (e.g., ATCC Deposit No. CCL-86), which are known to bemoderately susceptible (about 50% lysis under these conditions) to lysisby NK-92 cells, were used for most purposes, including verification ofthe cytotoxicity of the effector cells.

Approximately 2×10⁶ of the selected target cells were washed bysuspension in RPMI medium and sedimentation at 1300 rpm for 5 minutes.After removal of the supernate, 20 μL of FBS and 100 μCi ofNa[⁵¹Cr]chromate was added and the cells incubated at 37° C. for 60-90minutes with mixing every 30 minutes. The labeled target cells werewashed three times by suspension in 10 mL of RPMI medium andsedimentation at 1500 rpm for 5 minutes. The final cell pellet wasre-suspended in α-MEM and diluted to a concentration of 1×10⁵/mL. Targetcells for use in redirected cytotoxicity or ADCC assays were furtherincubated with the appropriate antibody at a final concentration of0.01-5 μg/mL for 10-15 minutes at room temperature.

One-hundred μL of the selected type of target cells and 100 μL of theappropriate concentration of effector cells were added to each well of a96 well V-bottom plate. Three to six replicate wells were prepared ateach E:T ratio. At least 6 wells were allocated to each of a spontaneouslysis control (effector cells replaced with 100 μL of α-MEM) and totalrelease control (effector cells replaced with 100 μL of 2%t-Octylphenoxypolyethoxyethanol (Triton X-100®) detergent in α-MEM).Desirably, an additional six or more wells are allocated to the use ofunmodified NK-92 effector cells that do not express CD16 as a proceduralcontrol and internal standard. The plate was then centrifuged at 500 rpmfor 3 minutes and incubated for 4 hours at 37° C. in an atmosphere of 7%CO₂/balance air. At the end of the incubation period, the plate wascentrifuged at 1500 rpm for 8 minutes, and 100 μL of the supernate wascollected from each well for counting in a γ counter to measure of ⁵¹Crrelease. The percentage of specific target cell lysis was calculated as:

${\%\mspace{11mu}{specific}\mspace{14mu}{lysis}} = {\frac{\begin{matrix}\left( {{{mean}\mspace{14mu}{cpm}\mspace{14mu}{experimental}\mspace{14mu}{release}} -} \right. \\\left. {{mean}\mspace{14mu}{cpm}\mspace{14mu}{spontaneous}\mspace{14mu}{lysis}} \right)\end{matrix}}{\begin{matrix}\left( {{{mean}\mspace{14mu}{cpm}\mspace{14mu}{total}\mspace{14mu}{release}} -} \right. \\\left. {{mean}\mspace{14mu}{cpm}\mspace{14mu}{{spontaneous}.\;{release}}} \right)\end{matrix}} \cdot 100}$

Example 5 CD16-Mediated Cell Lysis

A monoclonal antibody specific for CD16 was used in redirectedcytotoxicity assays in those cases where a target cell line thatexpressed a Fc receptor other than CD16 was available. In particular,the FcgR+ mouse mastocytoma cell line P815 and human FcγR⁺ myelocyticcell line THP-1 were used as targets in combination with the anti-CD16monoclonal antibody (mAb) 3G8 (Fleit et al., 1982; Perussia andTrinchieri, 1984) to evaluate NK-92-CD16, NK-92-CD16-γ and NK-92-CD16-ζcells (FIGS. 3 and 4). In this format, the target cell receptor isthought to bind the Fc portion of the antibody, while the F(ab) portionof the antibody binds to CD16 on the effector cell. Alternatively,redirected cytotoxicity assays can be performed using target cells thatexpress a unique antigen, but which do not express a Fc receptor, inconjunction with a bi-specific antibody construct. In this format, oneF(ab) binding domain of the bi-specific antibody specifically binds thetarget cell antigen while another F(ab) domain specifically binds toCD16. The evaluation of NK-92-CD16, NK-92-CD16-γ or NK-92-CD16-ζ cellsin this format was carried out using SKOV-3 as target cells and thechimeric antibody 2B1 as the cross-linking agent. The 2B1 chimericbi-specific antibody has one binding domain that is specific forHER2/neu and a second binding domain that is specific for CD16 (Clark etal., 1997; Weiner et al., 1995a; Weiner et al., 1995b) (FIG. 5). FIG. 6illustrates the cytotoxicity of NK-92-CD16, NK-92-CD16-γ andNK-92-CD16-ζ cells against P815 target cells in a redirectedcytotoxicity assay using 2B1 chimeric antibody after performing a⁵¹Cr-release assay for four hours. This Figure shows that at less thansaturating antibody concentrations, cytotoxicity is a function of bothantibody concentration and the level of expression of CD16 on the NK-92effector cells.

The following procedure was used in performing both ADCC and redirectedcytotoxicity assays, the type of assay being determined by thecharacteristics of the target cell and selected antibody. The selectedtarget cells were labeled with Na[⁵¹Cr]chromate as described in Example4. Aliquots of the ⁵¹[Cr]-labeled target cells were further incubatedwith the selected antibody at multiple concentrations between 0.01 μgand 5 μg/mL for 15 minutes at room temperature, washed with α-MEM, andadjusted to a concentration of 1×10⁵ cells/mL before use. One-hundred μLof the selected type of target cells and 100 μL of effector cells atcell concentrations of 1×10⁵/mL (E:T=1:1), 5×10⁵/mL (E:T=5:1), 1×10⁶/mL(E:T=10:1), 2×10⁶/mL (E:T=20:1) or as appropriate to the determinationbeing performed were added to each well of a 96-well V-bottom plate.Three to six replicate wells were prepared at each E:T ratio to beevaluated. At least 6 wells were allocated to each of a spontaneouslysis control (effector cells replaced with 100 μL of α-MEM) and totalrelease control (effector cells replaced with 100 μL of 2% Triton X-100detergent in α-MEM). An additional three wells at each E:T ratio wereallocated to “non-ADCC” controls in which the target cells were notexposed to the antibody. Desirably, an additional 6 or more wells areallocated to the use of unmodified NK-92 effector cells that do notexpress CD16 as a procedural control and internal standard. The platewas then centrifuged at 500 rpm for 3 minutes and incubated for 4 hoursat 37° C. in an atmosphere of 7% CO₂/balance air. At the end of theincubation period, the plate was centrifuged at 1500 rpm for 8 minutesand 100 mL of the supernate was collected from each well for counting ina γ counter as a measure of ⁵¹[Cr] release due to cytotoxicity. Thepercentage of specific lysis was calculated as described in Example 4.

These assays can also employ NK-92-CD16 cells expressing varying surfacelevels of CD16 (through cell sorting or via γ or ζ co-transduction), aswell as NK-92 transduced with the high affinity polymorphic allele ofthe CD16 gene (F157V). These variants to the invention provide a broaddynamic range of assay sensitivities. Although this Example is describedwith reference to monoclonal antibodies and bi-specific antibodyconstructs, polyclonal antibodies and other types of antibody constructshaving the appropriate characteristics can also be used in the practiceof this invention.

Example 6 Screening and Evaluation of Therapeutic Antibodies

The selected target cells were labeled with Na[⁵¹Cr]chromate asdescribed in Example 4 and adjusted to a concentration of 1×10⁵ cells/mLbefore use. A 100 μL aliquot of labeled target cells was thentransferred to each well of the requisite number of 96-well plates. Theimmunoglobulin concentrations in the hybridoma supernates to be screenedwere optionally, but preferably, adjusted to a convenient nominalconcentration of 1 μg/mL. At least 100 μL aliquots of each hybridomasupernate was added to each of three target cell containing wells;incubated for 15 minutes at room temperature, washed with α-MEM, andre-suspended in 100 μL of α-MEM. The effector cell concentration wasadjusted as appropriate to achieve the desired E:T ratio in the assay.For example, if an E:T ratio of 10:1 was desired, the effector cellconcentration was adjusted to 1×10⁶ cells/mL. The assay was initiated byadding 100 μL of effector cells to each well. The plates were thencentrifuged at 500 RPM for 3 minutes and incubated for 4 hours at 37° C.in an atmosphere of 7% CO₂/balance air. At the end of the incubationperiod, the plate was centrifuged at 1500 rpm for 8 minutes and 100 μLof the supernate was collected from each well for counting in a γcounter as a measure of ⁵¹[Cr] release due to cytotoxicity. Thepercentage of specific lysis was calculated as described in Example 4.At least six wells were allocated to each of a spontaneous lysis control(effector cells replaced with 100 μL of α-MEM) and a total releasecontrol (effector cells replaced with 100 μL of 2% Triton X-10 detergentin α-MEM) on each plate. An additional six wells in each set of plateswas allocated to each of a “no antibody” control (target cells nottreated with antibody) and a NK-92 cytolysis control. Specific lysis wasreported as the average of three replicate wells after correction forthe appropriate controls. Efficacy can be likewise be assessed by themeasurement of surrogate indicators such as cytokine release by theNK-92-CD16 cells, the up-regulation of NK cell activation markers suchas CD25, CD69 and/or CD95L, activation of transcription factors, such asNF-AT or NF-κB within the NK-92 cells, or the activation of caspases orother markers of apoptosis in the target cells.

In most cases, relatively small numbers (often only one) of antibodyconstructs were prepared. In such cases, screening was not necessary,and the construct as more conveniently evaluated using a direct assay,such as described in Example 5. Similarly, the relatively fewpotentially useful antibodies detected during screening weresubsequently characterized in more detail using assays such as describedin Example 5. Varying concentrations of purified antibodies can betested to compare efficacies for inducing ADCC. Furthermore, comparativetesting of ADCC potential of antibodies on NK-92 cells bearing eitherlow affinity (F157) vs. higher affinity (F157V) forms of CD16 offered aconvenient, reproducible assay to address therapeutic efficacies ofindividual antibodies in the context of both of these known humanalleles of CD16. This also circumvented the need for the user toidentify specific donors that are homozygous for each of the two allelesfor such assays.

Example 7 CD16 Mediated Cytokine Production

Upon activation, NK-92 cells are known to produce and secrete cytokines,including interferon-γ (IFN-γ), tumor necrosis factor (TNF-α andothers), interleukins (IL)-5, -10 and -13, granulocyte-macrophagecolony-stimulating factor (GM-CSF), nitric oxide and others uponactivation. The production of these cytokines can be determined bystandard methods including cytokine-specific enzyme-linked immunosorbantassay (ELISA) kits that are available from multiple commercial sources(e.g., BD Pharmingen; San Diego, Calif.). The production of cytokines byNK-92, NK-92-CD16, NK-92-CD16-γ and NK-92-CD16-ζ cells in response toCD16 mediated stimulation can be determined in a manner that isanalogous to the ADCC and redirected cytotoxicity assays described inExamples 4 and 5.

Effector cells (NK-92, NK-92-CD16, NK-92-CD16-γ and NK-92-CD16-ζ) werewashed by suspension in α-MEM (without IL-2) and sedimentation at 1300rpm for 5 minutes. The cell pellet was suspended in α-MEM, the cellscounted, and aliquots prepared at cell concentrations of 1×10⁵/mL(E:T=1:1), 5×10⁵/mL (E:T 5:1), 1×10⁶/mL (E:T=10:1), 2×10⁶/mL (E:T=20:1),or as appropriate to the determination being performed.

The type of target cell used in these assays was selected on the basisof the requirements of the particular determination being performed.Raji cells, which are known to be moderately susceptible (about 50%lysis under these conditions) to lysis by NK-92 cells, were used formost purposes, including verification of the cytotoxicity of theeffector cells.

One hundred μL of varying concentrations of effector cells were combinedwith a constant concentration of antibody treated target cells (notlabeled with ⁵¹[Cr]) in wells of a 96-well V-bottom plate. Three to sixreplicate wells were prepared at each E:T ratio to be evaluated. Atleast 6 wells each were allocated as controls for non-CD16 specificeffector cell activation in which the target cells were replaced with100 mL of α-MEM (spontaneous release) or with 100 μL of 2% Triton X-100(total release). Additional controls using target cells that have notbeen antibody treated and target or effector cells that had been treatedwith F(ab′)₂ fragments to suppress non-CD16 specific effector cellactivation were also included as appropriate. Transduced NK-92 cellsexpressing different levels or affinities of CD16 could have been usedas additional controls in the manner previously described. The plate wascentrifuged at 500 rpm for 3 minutes and incubated for 4 hours at 37° C.in an atmosphere of 7% CO₂/balance air. At the end of the incubationperiod, the plate was centrifuged at 1500 rpm for 8 minutes, andaliquots of the supernate were collected from each well to quantifycytokine concentrations, using commercially available cytokine ELISAkits (e.g., BD Pharmingen; San Diego, Calif.). Effector cell cytokineproduction was generally determined to track effector cell cytotoxicityand could therefore be taken as an alternative indicator of effectorcell activation.

Example 8 NK-92 Cell Stimulation by IL-2

Certain cytokines, particularly IL-2, IL-12, IL-15 and IL-18, are knownto promote the growth, survival, cytotoxicity and cytokine releasingactivities of NK, NK-92, NK-92-CD16, NK-92-CD16-γ and NK-92-CD16-ζ, andother NK-92 variant cells both in-vitro and in-vivo. By way of example,the cells transduced in Examples 2 and 3 proliferated and exhibitedstable levels of CD16 expression, cytotoxicity and cytokine response forseveral months without the need for antibiotic selection whensub-cultured with fresh IL-2-containing medium every 4 days. Conversely,when these same cells were passed without the addition of IL-2, theyexhibited cytotoxicity and cytokine production levels that declined withtime through the 4-day culture period and returned to higher levels onthe first day after fresh IL-2 addition. Furthermore, cells maintainedin the absence of IL-2 specifically lysed a narrower range of cell typesthan did cells maintained in the presence of IL-2. This behavior oftransduced NK-92 cells and derivatives closely reflects that ofunmodified primary NK cells. For these reasons, it is desirable to assaycells and transduced derivatives at consistent intervals after passagewith defined concentrations of IL-2. Similarly, it is desirable toco-administer IL-2 when these cells are being used for in-vivotherapeutic purposes. In those cases where the provision of exogenousIL-2 is inconvenient or otherwise undesirable, the NK-92 ml or NK-92cicell line that has been engineered to express endogenous IL-2 at levelsthat promote NK-92 proliferation, survival and activity can also beemployed. The NK-92 ml, NK-92ci and other NK-92 derived cell lines canbe transduced in the same manners as described for the parent NK-92 cellline in Examples 2 and 3.

From the foregoing, it will be observed that numerous variations andmodifications can be effected without departing from the spirit andscope of the novel concept of the invention. It is to be understood thatno limitation with respect to the specific methods and apparatusillustrated herein is intended or should be inferred. It is, of course,intended to cover by the appended claims all such modifications as fallwithin the scope of the claims.

TABLE Z Table of Abbreviations Abbreviation Definition ADCCAntibody-dependent cellular cytotoxicity CD Cluster of determination CTLCytotoxic T-lymphocytes DNA Deoxyribonucleic acid EGFP Enhanced greenfluorescent protein ELISA Enzyme-linked immunosorbent assay ErbB2Proto-oncogene that encodes a membrane- bound receptor tyrosine kinaseof the epithelial growth factor receptor (EGFR) family; also known asHER-2/neu FBS Fetal bovine serum Fc Denotes constant region of anantibody FHS Fetal horse serum GM-CSF Granulocyte-macrophagecolony-stimulating factor IFN Interferon IL Interleukin MHC-I Majorhistocompatibility complex class I MEM Minimum essential medium MHCMajor histocompatibility complex NF-AT nuclear factor of activatedT-cells NF-κB Nuclear factor-κB NK Natural killer PBL Peripheral bloodlymphocyte RAG recombinase activating gene RhIL Recombinant, humaninterleukin RNA Ribonucleic acid Rpm Rotations per minute RPMI RoswellPark Memorial Institute SCID Severe combined immunodeficiency TNF Tumornecrosis factor TriBi trimeric, bi-specific

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1. A modified NK-92 cell available from American Type Culture Collectionas Deposit No. PTA-8837.